Kits for detecting ribonucleic acid

ABSTRACT

Compositions, methods, and kits for detecting one or more species of RNA molecules are disclosed. In one embodiment, a first adaptor and a second adaptor are ligated to the RNA molecule using a polypeptide comprising double-strand specific RNA ligase activity, without an intervening purification step. The ligated product is reverse transcribed, then at least some of the ribonucleosides in the reverse transcription product are removed. Primers are added and amplified products are generated. In certain embodiments, the sequence of at least part of at least one species of amplified product is determined and at least part of the corresponding RNA molecule is determined. In some embodiments, at least some of the amplified product species are detected, directly or indirectly, allowing the presence and/or quantity of the RNA molecule of interest to be determined.

CROSS-RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.13/463,758 filed May 3, 2012, which is a continuation of U.S.application Ser. No. 12/835,869 filed Jul. 14, 2010 (now U.S. Pat. No.8,192,941), which is a continuation of, and claims priority to,International Patent Application No. PCT/US2009/030822, having aninternational filing date of Jan. 13, 2009, which application claims thebenefit of U.S. Provisional Application No. 61/020,913 filed Jan. 14,2008, U.S. Provisional Application No. 61/039,460 filed Mar. 26, 2008and U.S. Application No. 61/047,549 filed Apr. 24, 2008. Eachapplication is incorporated by reference herein in its entirety.

FIELD

The present teachings generally relate to methods, reagents, and kitsfor detecting, amplifying, and quantifying ribonucleic acid (RNA),including but not limited to coding RNA and non-coding RNA (ncRNA).

INTRODUCTION

Analysis of genome expression patterns provides valuable insight intothe role of differential expression in a wide variety of biologicalprocesses, including but not limited to, various disease states. Suchanalysis, whether mRNA-based gene expression or small non-codingRNA-based expression analysis, is becoming a rapidly expanding avenue ofinvestigation in many disciplines in the biological sciences. Smallnon-coding RNA discovery is also an area of great scientific and medicalinterest. It is believed that by knowing what parts of the genome aretranscribed when and why, a better understanding of many complex andinter-related biological processes may be obtained.

Small non-coding RNAs are rapidly emerging as significant effectors ofgene regulation in a multitude of organisms spanning the evolutionaryspectrum. Animals, plants and fungi contain several distinct classes ofsmall RNAs; including without limitation, miRNAs, siRNAs, piRNAs, andrasiRNAs. These tiny gene expression modulators typically fall withinthe size range of ˜18-40 nt in length, however their effect on cellularprocesses is profound. They have been shown to play critical roles indevelopmental timing and cell fate mechanisms, tumor progression,neurogenesis, transposon silencing, viral defense and many more. Theyfunction in gene regulation by binding to their targets and negativelyeffecting gene expression by a variety of mechanisms includingheterochromatin modification, translational inhibition, mRNA decay andeven nascent peptide turnover mechanisms. Therefore, identification ofthe small RNAs in a given sample can greatly facilitate gene expressionanalysis.

Some small RNAs are produced from defined locations within the genome.MicroRNAs are such a class; they are typically transcribed by RNApolymerase II from polycistronic gene clusters or can also be generatedfrom pre-mRNA introns. Thus far several thousand unique miRNA sequencesare known. Other classes of small RNAs, such as piRNAs or endogenoussiRNA, are not typically transcribed from a defined locus in the genome.Instead, they are generated in response to events such as viralinfections or retrotransposon expression and serve to silence these‘foreign’ sequences that would otherwise result in serious detriment tothe cell. Descriptions of ncRNA can be found in, among other places,Eddy, Nat. Rev. Genet. 2:919-29, 2001; Mattick and Makunin, Human Mol.Genet. 15:R17-29, 2006; Hannon et al., Cold Springs Harbor Sympos.Quant. Biol. LXXI:551-64, 2006. Sequencing the entire population ofsmall RNAs in a sample provides a direct method to identify and evenprofile all classes of these RNAs at one time.

SUMMARY

The present teachings are directed to methods, reagents, and kits fordetecting and quantitating: (i) small RNA molecules, also referred to asuntranslated functional RNA, non-coding RNA (ncRNA), and smallnon-messenger RNA (snmRNA); and (ii) coding RNA, which may or may not befragmented and/or fractionated by methods known in the art.

According to certain disclosed methods, a ligation reaction compositionis formed comprising at least one RNA molecule to be detected, at leastone first adaptor, at least one second adaptor, and a double-strandspecific RNA ligase. The first adaptor comprises a first oligonucleotidecomprising at least two ribonucleosides on the 3′-end and a secondoligonucleotide that comprises a single-stranded portion when the firstoligonucleotide and the second oligonucleotide are hybridized together.The second adaptor comprises a third oligonucleotide that comprises a 5′phosphate group and a fourth oligonucleotide that comprises asingle-stranded portion when the third oligonucleotide and the fourtholigonucleotide are hybridized together. A first adaptor and a secondadaptor are ligated to an RNA molecule in the ligation reactioncomposition by the double-strand specific RNA ligase to form a ligatedproduct. The first adaptor and the second adaptor anneal with the RNAmolecule in a directional manner due to their structure and each adaptoris ligated simultaneously or nearly simultaneously to the RNA moleculewith which it is annealed, rather than sequentially (for example, when asecond adaptor and the RNA molecule are combined with a ligase and thesecond adaptor is ligated to the 3′ end of the RNA molecule, thensubsequently a first adaptor is combined with the ligated RNAmolecule-second adaptor and the first adaptor is then ligated to the 5′end of the RNA molecule-second adaptor, with an intervening purificationstep between ligating the second adaptor to the RNA molecule andligating the first adaptor to the RNA molecule, see, e.g., Elbashir etal, Genes and Development 15: 188-200, 2001; Berezikov et al., Nat.Genet. Supp. 38: S2-S7, 2006). It is to be appreciated that the order inwhich components are added to the ligation reaction composition is notlimiting and that the components may be added in any order. It is alsoto be appreciated that during the process of adding components, anadaptor may be ligated with a corresponding RNA molecule in the presenceof a ligase before all of the components of the reaction composition areadded, for example but without limitation, a second adaptor may beligated with a corresponding RNA molecule in the presence of a ligasebefore the first adaptors are added, and that such reactions are withinthe intended scope of the current teachings, provided there is not apurification procedure between the time one adaptor is ligated to theRNA molecule and the time the other adaptor is ligated to the RNAmolecule. An RNA-directed DNA polymerase (sometimes referred to as anRNA-dependent DNA polymerase) is combined with the ligated product toform reaction mixture, which is incubated under conditions suitable fora reverse transcribed product. The reverse transcribed product iscombined with a ribonuclease, typically ribonuclease H(RNase H), and atleast some of the ribonucleosides are digested from the reversetranscribed product to form an amplification template.

The amplification template is combined with at least one forward primer,at least one reverse primer, and a DNA-directed DNA polymerase(sometimes referred to as a DNA-dependent DNA polymerase) to form anamplification reaction composition. The amplification reactioncomposition is thermocycled under conditions suitable to allow amplifiedproducts to be generated. In some embodiments, at least one species ofamplified product is detected. In some embodiments, a reporter probeand/or a nucleic acid dye is used to indirectly detect the presence ofat least one of the RNA species in the sample. In certain embodiments,an amplification reaction composition further comprises a reporterprobe, for example but not limited to a TaqMan® probe, molecular beacon,Scorpion™ primer or the like, or a nucleic acid dye, for example but notlimited to, SYBR® Green or other nucleic acid binding dye or nucleicacid intercalating dye. In certain embodiments of the current teachings,detecting comprises a real-time or end-point detection technique,including without limitation, quantitative PCR. In some embodiments, thesequence of at least part of the amplified product is determined, whichallows the corresponding RNA molecule to be identified. In someembodiments, a library of amplified products comprising alibrary-specific nucleotide sequence is generated from the RNA moleculesin a starting material, wherein at least some of the amplified productspecies share a library-specific identifier, for example but not limitedto a library-specific nucleotide sequence, including without limitation,a barcode sequence or a hybridization tag, or a common marker oraffinity tag. In some embodiments, two or more libraries are combinedand analyzed, then the results are deconvoluted based on thelibrary-specific identifier.

According to certain disclosed methods, only one polymerase, a DNApolymerase comprising both DNA-directed DNA polymerase activity andRNA-directed DNA polymerase activity, is employed in the reversetranscription reaction composition and no additional polymerase is used.In other method embodiments, both an RNA-directed DNA polymerase and aDNA-directed DNA polymerase are added to the reverse transcriptionreaction composition and no additional polymerase is added to theamplification reaction composition.

In some embodiments, a method for detecting a RNA molecule in a samplecomprises combining the sample with at least one first adaptor, at leastone second adaptor, and a polypeptide comprising double-strand specificRNA ligase activity to form a ligation reaction composition in which theat least one first adaptor and the at least one second adaptor areligated to the RNA molecule of the sample to form a ligated product inthe same ligation reaction composition, and detecting the RNA moleculeof the ligated product or a surrogate thereof. In some embodiments, theat least one first adaptor comprises a first oligonucleotide having alength of 10 to 60 nucleotides and comprising at least tworibonucleosides on the 3′-end, and a second oligonucleotide comprising anucleotide sequence substantially complementary to the firstoligonucleotide and further comprising a single-stranded 5′ portion of 1to 8 nucleotides when the first oligonucleotide and the secondoligonucleotide are duplexed. In some embodiments, the at least onesecond adaptor comprises a third oligonucleotide having a length of 10to 60 nucleotides and comprising a 5′ phosphate group, and a fourtholigonucleotide comprising a nucleotide sequence substantiallycomplementary to the third oligonucleotide and further comprising asingle-stranded 3′ portion of 1 to 8 nucleotides when the thirdoligonucleotide and the fourth oligonucleotide are duplexed. In someembodiments, the single-stranded portions independently have adegenerate nucleotide sequence, or a sequence that is complementary to aportion of the RNA molecule. In some embodiments, the first and thirdoligonucleotides have a different nucleotide sequence. In the ligatonreaction composition, the RNA molecule to be detected hybridizes withthe single-stranded portion of the at least one first adaptor and thesingle-stranded portion of the at least one second adaptor.

In some embodiments, detecting the RNA molecule or a surrogate thereofcomprises combining the ligated product with i) a RNA-directed DNApolymerase, ii) a DNA polymerase comprising DNA dependent DNA polymeraseactivity and RNA dependent DNA polymerase activity, or iii) aRNA-directed DNA polymerase and a DNA-directed DNA polymerase; reversetranscribing the ligated product to form a reverse transcribed product;digesting at least some of the ribonucleosides from the reversetranscribed product with ribonuclease H to form an amplificationtemplate; combining the amplification template with at least one forwardprimer, at least one reverse primer, and a DNA-directed DNA polymerasewhen the ligated product is combined as in i), to form an amplificationreaction composition; cycling the amplification reaction composition toform at least one amplified product, and determining the sequence of atleast part of the amplified product, thereby detecting the RNA molecule.

In some embodiments, a method for generating an RNA library comprisescombining a multiplicity of different RNA molecules with a multiplicityof first adaptor species, a multiplicity of second adaptor species, anda double-strand specific RNA ligase to form a ligation reactioncomposition, wherein the at least one first adaptor comprises a firstoligonucleotide comprising at least two ribonucleosides on the 3′-endand a second oligonucleotide that comprises a single-stranded portionwhen the first oligonucleotide and the second oligonucleotide arehybridized together, and wherein the at least one second adaptorcomprises a third oligonucleotide that comprises a 5′ phosphate groupand a fourth oligonucleotide that comprises a single-stranded portionwhen the third oligonucleotide and the fourth oligonucleotide arehybridized together and ligating the at least one first adaptor and theat least one second adaptor to the RNA molecule to form a multiplicityof different ligated product species, wherein the first adaptor and thesecond adaptor are ligated to the RNA molecule in the same ligationreaction composition. The method further comprises combining themultiplicity of ligated product species with an RNA-directed DNApolymerase, reverse transcribing at least some of the multiplicity ofligated product species to form a multiplicity of reverse transcribedproduct species, digesting at least some of the ribonucleosides from atleast some of the multiplicity of reverse transcribed products with aribonuclease H(RNase H) to form a multiplicity of amplification templatespecies, combining the multiplicity of amplification template specieswith at least one forward primer, at least one reverse primer, and aDNA-directed DNA polymerase to form an amplification reactioncomposition, and cycling the amplification reaction composition to forma library comprising a multiplicity of amplified product species,wherein at least some of the amplified product species comprise anidentification sequence that is common to at least some of the otheramplified product species in the library.

Kits for performing certain of the instant methods are also disclosed.These and other features of the present teachings are set forth herein.

DRAWINGS

The skilled artisan will understand that the drawings, described below,are for illustration purposes only. These figures are not intended tolimit the scope of the present teachings in any way.

FIG. 1 provides a schematic overview of various exemplary methodembodiments of the current teachings.

FIG. 2A-FIG. 2B: FIG. 2A schematically depicts an exemplary firstadaptor 21 and an exemplary second adaptor 22; FIG. 2B schematicallydepicts the exemplary first and second adaptors shown in FIG. 1Adirectionally annealed to an exemplary RNA molecule 23. The ligationjunction for the first adaptor 21 and the 5′ end of the RNA molecule 23is shown by arrow 24 and the ligation junction for the second adaptor 22and the 3′ end of the RNA molecule 23 is shown by arrow 25. Openrectangles depict RNA sequence such as for 21A and 23. Horizontal solidlines depict DNA sequence such as for 21B and 22A.

FIG. 3 provides a schematic overview of an exemplary embodiment of thecurrent teachings. A population of small RNA molecules 33 is combinedwith a first adaptor 31 and a second adaptor 32 and Rnl2 ligase to formligated product 34. Unannealed adaptors and/or undesired annealedbyproduct molecules 35 may also be present in the ligated reactioncomposition. The reaction composition is combined with an RNA-directedDNA polymerase to generate reverse transcribed product 36, whichcomposition is then combined with ribonuclease H to generateamplification template 38. The amplification template 38 is combinedwith a DNA-directed DNA polymerase, a forward primer 310 and a reverseprimer 311 to form an amplification reaction composition. In thisillustrative embodiment, the reverse primer further comprises anidentification sequence 312, sometimes referred to as a “bar code”sequence.

FIG. 4 provides a schematic overview of an exemplary embodiment of thecurrent teachings. In this illustrative embodiment, the amplifiedproduct is gel purified (Gel Purif.) and comprises an insert sequence(shown by a curved bracket in FIG. 4), a first primer region (shown asP1 in FIG. 4), and a second primer region (shown as P2 in FIG. 4) thatincludes a bar code or identification sequence (shown as be in FIG. 4).

FIG. 5 depicts an electropherogram of the exemplary amplified productsgenerated as described in Example 1.

Lane 1: 10 bp DNA ladder (100 ng; Invitrogen P/N 10821-015, Carlsbad,Calif.);

Lane 2: starting material was 100 ng total RNA, minus ligase control;

Lane 3: starting material was 100 ng total RNA, minus RT control;

Lane 4: starting material was 100 fmol mirVana™ Reference Panel v.9.1(Applied Biosystems P/N 4388891, Foster City, Calif.);

Lane 5: starting material was 100 ng total RNA; and

Lane 6: starting material was flashPAGE™ Fractionator System-purifiedRNA from 5 μg total RNA.

FIG. 6 depicts an electropherogram of the exemplary amplified productsgenerated using various double strand-dependent ligases, alone or incombination, as described in Example 2.

Lane 1: 100 ng 10 bp DNA ladder (Invitrogen);

Lane 2: 10 units of bacteriophage T4 RNA ligase 2, 200 U reversetranscriptase (RT);

Lane 3: 10 units of bacteriophage T4 RNA ligase 2, no RT;

Lane 4: 10 units bacteriophage T4 RNA ligase 1,200 U RT;

Lane 5: 10 units bacteriophage T4 RNA ligase 1, no RT;

Lane 6: 10 units bacteriophage T4 DNA ligase, 200 U RT;

Lane 7: 10 units bacteriophage T4 DNA ligase, no RT;

Lane 8: 5 units each of bacteriophage T4 RNA ligase I and bacteriophageT4 DNA ligase, 200 U RT;

Lane 9: 5 units each of bacteriophage T4 RNA ligase I and bacteriophageT4 DNA ligase, no RT; and

Lane 10: no ligase, 200 U RT.

FIG. 7A depicts an electropherogram of exemplary ligated products (shownby arrow annotated double ligation) generated in a ligation reactioncomposition comprising various first adaptors, various second adaptors,or combinations of various first adaptors and various second adaptors,with a multiplicity of different miRNA molecules comprised ofapproximately equimolar concentrations of about five hundred differentspecies of synthetic miRNA molecules and RNA ligase 2 of bacteriophageT4, as described in Example 4. The numbers 4, 6, and 8 across the top ofFIG. 7A correspond to the number of degenerate nucleotide sequences onthe second oligonucleotide of each of the first adaptors and the fourtholigonucleotide of each of the second adaptors in the reactioncomposition (shown as N in FIG. 7B).

X-axis:

T3-4 indicates that the corresponding ligation products were generatedin a ligation composition comprising only first adaptors comprising thestructure T3:27 N (see FIG. 7B), where in this case N equals 4degenerate nucleotides;

T3-6 indicates that the corresponding ligation products were generatedin a ligation reaction composition containing only first adaptorscomprising the structure T3:27 N, where N in this case equals 6degenerate nucleotides;

T3-8 indicates that the corresponding ligation products were generatedin a ligation reaction containing only first adaptors comprising thestructure T3:27 N, where N in this case equals 8 degenerate nucleotides;

T7-4 indicates that the corresponding ligation products were generatedin a ligation reaction with only second adaptors comprising thestructure T7:N 28 (see FIG. 7B), where N in this case equals 4degenerate nucleotides;

T7-6 indicates that the corresponding ligation products were generatedin a ligation reaction with only second adaptors comprising thestructure T7:N 28 (see FIG. 7B), where N in this case equals 6degenerate nucleotides;

T7-8 indicates that the corresponding ligation products were generatedin a ligation reaction with only second adaptors comprising thestructure T7:N 28, where N in this case equals 8 degenerate nucleotides;

T3-4+T7-4 indicates that the corresponding ligation products weregenerated in a ligation reaction with first adaptors comprising thestructure T3:27 N and second adaptors comprising the structure T7:N 28,where N in this case equals 4 degenerate nucleotides in all species ofboth adaptors;

T3-6+T7-6 indicates that the corresponding ligation products weregenerated in a ligation reaction with first adaptors comprising thestructure T3:27 N and second adaptors comprising the structure T7:N 28,where N in this case equals 6 degenerate nucleotides in all species ofboth adaptors; and

T3-8+T7-8 indicates that the corresponding ligation products weregenerated in a ligation reaction with first adaptors comprising thestructure T3:27 N and second adaptors comprising the structure T7:N 28,where N in this case equals 8 degenerate nucleotides in all species ofboth adaptors.

The number 27 refers to the length of the first oligonucleotide of thefirst adaptor and the number 28 refers to the length of the thirdnucleotide of the second adaptor.

FIG. 7B schematically depicts exemplary first and second adaptors of thecurrent teachings, where N represents a series of degenerate nucleosideson the lower strand of either the exemplary first adaptor or the secondadaptor, i.e., the second oligonucleotide or the fourth oligonucleotide,respectively.

FIG. 8A: depicts an electropherogram of exemplary ligated products(indicated by arrow) generated using various first adaptors, varioussecond adaptors, or combinations of various first adaptors and varioussecond adaptors, as described in Example 5 and depicted in FIG. 8B.

X-axis:

T3r2-6 indicates that the corresponding ligation products were generatedin a ligation reaction with only first adaptors comprising T3r2:27 6N(see FIG. 8B), where 6N equals six degenerate nucleotides and r2designates two 3′ ribonucleotides;

T7-6 indicates that the corresponding ligation products were generatedin a ligation reaction with only second adaptors comprising T7:6 N 28(see FIG. 8B), where 6N equals six degenerate nucleotides;

T3r2-6+T7-6 indicates that the corresponding ligation products weregenerated in a ligation reaction with first adaptors comprising T3r2:276N and second adaptors comprising T7: 6N 28, where and r2 designates two3′ ribonucleotides and where 6N equals six degenerate nucleotides in allspecies of both adaptors;

rT3-6 indicates that the corresponding ligation products were generatedin a ligation reaction with only first adaptors comprising rT3:27 6N,where 6N equals six degenerate nucleotides and rT3 designates allribonucleotides,

rT7-6 indicates that the corresponding ligation products were generatedin a ligation reaction with only second adaptors comprising rT7:6 N 28,where rT7 designates all ribonucleotides, 6N equals six degeneratenucleotides; and

rT3-6+rT7-6 indicates that the corresponding ligated products weregenerated in a ligation reaction with first adaptors comprising rT3:276N and second adaptors comprising rT7:6 N 28, where rT3 and rT7designate all ribonucleotides and 6N equals six degenerate nucleotidesin all species of both adaptors.

FIG. 8B schematically depicts two exemplary sets of first and secondadaptors of the current teachings (the two sets comprise (i)_(r)T3:27 6N(top first adaptor) and rT7:6 N 28 (top second adaptor) and (ii) T3r2:276N (bottom first adaptor) and T7:6 N 28 (bottom second adaptor), where6N represents a series of six degenerate nucleosides on the lower strandof the exemplary first adaptors and the lower strand of the exemplarysecond adaptors.

FIG. 9A-FIG. 9C. FIG. 9A and FIG. 9B depict electropherograms ofexemplary ligated products generated according to certain embodiments ofthe current teachings as described in Example 6. Three differentcombinations of first adaptors and second adaptors were tested fordouble ligation efficiency. These combinations included first adaptorsand second adaptors with both DNA upper strands (i.e., first and thirdoligonucleotides) except for two ribonucleosides on the 3′ end of thefirst oligonucleotide, both RNA upper strands (i.e., first and thirdoligonucleotides), or RNA upper strand on 5′ (first) adaptor (i.e.,first oligonucleotide) and DNA upper strand on 3′ (second) adaptor(i.e., third oligonucleotide). FIG. 9C provides a schematic of thelatter adaptor structure embodiment having exemplary first adaptors(rT3: 27 6N) and second adaptors (T7:6 N 28) (individually alsodescribed in FIG. 8B).

FIG. 10A and FIG. 10B depict two electropherograms showing exemplaryligation products generated according to certain embodiments of thecurrent teachings using a series of ligation reaction compositions, eachcomprising (1) Rnl2 ligase, (ii) a pool of synthetic miRNA molecules(mirVana miRNA Reference Panel v 9.1, P/N 4388891 (Ambion, Austin, Tex.;described herein) at a concentration of 2 and 0.2 picomoles (pmol), and(iii) first and second adaptors as disclosed herein at upper to lowerstrand ratios of 10/50, 5/25, 1/5, 1/50, 5/50, 10/50, 25/50, 5/100 or5/500 (upper strand/lower strand) as shown and described in Example 6.

FIG. 11 schematically depicts certain embodiments of the currentteachings wherein various subpopulations of nucleic acid are removedand/or purified from the sample. Embodiments of the methods can be usedfor small RNA detection and isolation and for whole transcriptomesequencing.

FIG. 12 depicts a graph of log₂ fold change (FC) in −ΔΔCT determinedusing an exemplary TaqMan®-based detection of illustrative amplifiedproducts generated according to one method of the current teachings(shown on y-axis as Log 2 (FC) TaqMan (−ddCt)) versus the Log 2 (FC)determined using an exemplary sequencing detection technique using theSOLiD™ Sequencing System with an aliquot of the same illustrativeamplified products (shown on x-axis as Log 2 (FC SOLiD™) as provided byExample 7.

FIG. 13A and FIG. 13B depict electropherograms comprising exemplaryamplified products generated by certain embodiments of the currentteachings visualized using SYBR® Gold staining as described in Example7.

FIG. 14 schematically depicts various embodiments of the currentteachings comprising detecting RNA molecules of interest by quantitatingexemplary amplified products generated according to the currentteachings using an intercalating dye, SYBR® Green (e.g., Example 8), fordetection in a real-time PCR reaction or SYBR® Gold staining ofelectrophoretically separated amplified products (“SYBR® Assay”).LEGenD: Ligase Enhanced Gene Detection refers to use of double-stranddependent ligase for assays as provided herein.

FIG. 15 schematically depicts an amplified product of the currentteachings as described in Example 8. P1 refers to a portion of theforward PCR primer. P2 refers to a portion of the reverse PCR primer.

FIG. 16A and FIG. 16B depict illustrative plots of real-time PCRdetected RNA molecules, either added to exemplary samples (FIG. 16A) ortwo ncRNA molecules present in the sample (endogenous miRNAs miR-16(FIG. 16B, top curve with diamond symbols) and miR-21 (FIG. 16B, bottomcurve with square symbols), as described in Example 8. The slope andy-intercepts for FIG. 16A are: SIC 34 (circles, top line):y=−3.2568x+32.916, R²=0.9918; SIC 8 (triangles): y=−3.4116x+29.444,R²=0.9886; SIC 37 (squares): y=−2.8517x+23.685, R²=0.9935; and SIC 36(diamonds, bottom line): y=−3.0381x+19.587, R²=0.999.

FIG. 17 provides an overview of a SOLiD™ Small RNA Expression Kitprocedure for generating a small RNA library as provided in Example 11.Size-selected amplified small RNA enters the SOLiD™ emulsion PCRprocedure at the “templated bead preparation” stage.

DESCRIPTION OF EXEMPLARY EMBODIMENTS

It is to be understood that both the foregoing general description andthe following detailed description are exemplary and explanatory onlyand are not intended to limit the scope of the current teachings. Inthis application, the use of the singular includes the plural unlessspecifically stated otherwise. For example, “a forward primer” meansthat more than one forward primer can be present; for example, one ormore copies of a particular forward primer species, as well as one ormore different forward primer species. Also, the use of “comprise”,“contain”, and “include”, or modifications of those root words, forexample but not limited to, “comprises”, “contained”, and “including”,are not intended to be limiting. The term “and/or” means that the termsbefore and after can be taken together or separately. For illustrationpurposes, but not as a limitation, “X and/or Y” can mean “X” or “Y” or“X and Y”.

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the described subject matter inany way. All literature and similar materials cited in this application,including patents, patent applications, articles, books, and treatisesare expressly incorporated by reference in their entirety for anypurpose. In the event that one or more of the incorporated literatureand similar materials defines or uses a term in such a way that itcontradicts that term's definition in this specification, thisspecification controls. While the present teachings are described inconjunction with various embodiments, it is not intended that thepresent teachings be limited to such embodiments. On the contrary, thepresent teachings encompass various alternatives, modifications, andequivalents, as will be appreciated by those of skill in the art.

The term “or combinations thereof” as used herein refers to allpermutations and combinations of the listed items preceding the term.For example, “A, B, C, or combinations thereof” is intended to includeat least one of: A, B, C, AB, AC, BC, or ABC, and if order is importantin a particular context, also BA, CA, CB, ACB, CBA, BCA, BAC, or CAB.Continuing with this example, expressly included are combinations thatcontain repeats of one or more item or term, such as BB, AAA, AAB, BBC,AAABCCCC, CBBAAA, CABABB, and so forth. The skilled artisan willunderstand that typically there is no limit on the number of items orterms in any combination, unless otherwise apparent from the context.

According to certain disclosed methods, for example but not limited to,the exemplary embodiment shown in FIG. 1, a ligation reactioncomposition is formed comprising at least one RNA molecule to bedetected, at least one first adaptor, at least one second adaptor, and adouble-strand specific RNA ligase (shown as Hybridization of First andSecond Adaptors to RNA Molecule). Typically, the starting materialcomprises a multiplicity of RNA species and multiplicities of differentfirst adaptors and different second adaptors.

As shown in FIG. 2A and FIG. 2B, the at least one first adaptor 21comprises a first oligonucleotide 21A comprising at least tworibonucleosides on the 3′-end and a second oligonucleotide 21B thatcomprises a single-stranded 5′ portion 21C when the firstoligonucleotide 21A and the second oligonucleotide 21B are hybridizedtogether (FIG. 2A), and wherein the at least one second adaptor 22comprises a third oligonucleotide 22A that comprises a 5′ phosphategroup (shown as “P” in FIG. 2B) and a fourth oligonucleotide 22B thatcomprises a single-stranded 3′ portion 22C when the thirdoligonucleotide 22A and the fourth oligonucleotide 22B are hybridizedtogether (FIG. 2A). It is to be appreciated that in this illustrativeembodiment, all of the nucleosides in the first oligonucleotide areribonucleosides, but that in other embodiments as many as all and as fewas two of the nucleosides of the first oligonucleotide can beribonucleosides, provided that the two 3′-most nucleosides of the firstoligonucleotide 21A are ribonucleosides; the remainder of the firstoligonucleotide may comprise ribonucleosides, deoxyribonucleosides, or acombination of both.

The single-stranded portions of the illustrative second and fourtholigonucleotides (21C of 21B and 22C of 22B, respectively) of FIG. 2Aare depicted as degenerate hexamer sequences (shown as NNNNNN). However,the use of first and/or second adaptors with sequence-specific singlestranded portions and also longer and shorter single-stranded portionsis within the scope of the current teachings. In some embodiments, thedegenerate sequences are deoxyribonucleotides. In some embodiments, thelength of the degenerate sequences is 4, 6, or 8 nucleotides. In someembodiments, the first oligonucleotide comprises ribonucleosides and thesecond, third and fourth oligonucleotides comprise deoxyribonucleotides.

First and second oligonucleotides are designed to be substantiallycomplementary with the exception of the single stranded portion 21C. Thesubstantially complementary portion can have a length of 10 to 60nucleotides. When annealed or duplexed to form a first adaptor, thefirst and second oligonucleotides can have one blunt end (as in FIG. 2Aand FIG. 2B) or can have an overhang of 1, 2, or 3 nucleotides at theend opposite the end having a single stranded portion. The overhang maybe on either the first or the second oligonucleotide.

Third and fourth oligonucleotides are designed to be substantiallycomplementary with the exception of the single stranded portion 22C. Thesubstantially complementary portion can have a length of 10 to 60nucleotides. When annealed or duplexed to form a second adaptor, thethird and fourth oligonucleotides can have one blunt end (as in FIG. 2Aand FIG. 2B) or can have an overhang of 1, 2, or 3 nucleotides at theend opposite the end having a single stranded portion. The overhang maybe on either the first or the second oligonucleotide.

Returning to FIG. 1, the ligation reaction composition is incubatedunder conditions suitable for a first adaptor and a second adaptor toanneal with an RNA molecule. The first and third oligonucleotides(“upper strands”) may be present in a 1:1 to 1:10 molar ratio to thesecond and fourth oligonucleotides (“lower strands”). In someembodiments, the molar ratio of “upper strands” to “lower strands” is1:5 or 1:2. A polypeptide comprising double-strand specific RNA ligaseactivity is used to ligate the annealed first adaptor-RNAmolecule-second adaptor complex to form a ligated product (shown as“Ligation” in FIG. 1). The first adaptor and the second adaptor areligated to the RNA molecule in the same reaction composition, ratherthan as two separate sequential ligation reactions with one or moreintervening separation or purification steps between ligating the twoadaptors to the RNA molecule. It is to be understood the order in whichthe components of the ligation reaction composition are added and thesequence in which the two adaptors are ligated to the RNA molecule aretypically not limitations of the current teachings provided.

As shown in FIG. 2B, in certain embodiments, the first and secondadaptors are hybridized to the RNA molecule such that (i) the 3′ end ofthe first oligonucleotide of a first adaptor and the 5′ end of the RNAmolecule are adjacently annealed to form a first ligation junction (forexample, 24 in FIG. 2B) (due to complementarity between the RNA moleculeand the single stranded portion of the first adaptor) and (ii) the 5′end of third oligonucleotide of a second adaptor and 3′ end of the sameRNA molecule are adjacently annealed to form a second ligation junction(for example, 25 in FIG. 2B) (due to complementarity between the RNAmolecule and the single stranded portion of the second adaptor), whereinthe first and the second ligation junctions are both suitable forligation using a polypeptide comprising double strand-specific RNAligase activity. In some embodiments, the polypeptide comprising doublestrand-specific RNA ligase activity comprises an Rnl2 family ligase,including without limitation, Rnl2 ligase.

An RNA-directed DNA polymerase is combined with the ligated product,along with suitable nucleotide triphosphates and a buffer solutioncomprising appropriate salts. This reaction mixture is incubated underconditions suitable for a reverse transcribed product to be generatedusing the ligated product as the template (shown as “ReverseTranscription” in FIG. 1). A separate reverse transcription primer isnot needed since the fourth oligonucleotide serves as the RT primer.

In some embodiments, the reverse transcribed product is placed on anarray and detected using standard methods known by one of skill in theart. In some embodiments, the reverse transcribed product is labeledwith biotin and detection is by using streptavidin binding thereto. Insome embodiments, the reverse transcribed product is purified usingglass fiber filters, beads or is gel-purified. In some embodiments, thereverse transcribed product is combined with a peptide comprisingribonuclease activity to form a digestion reaction composition andincubated under conditions suitable for digesting at least some of theribonucleosides from the reverse transcribed product to form anamplification template. In some embodiments, the peptide comprisingribonuclease activity comprises ribonuclease H(RNase H) activity (shownas “RNase H Digestion” in FIG. 1).

The amplification template is combined with at least one forward primer,at least one reverse primer, and a peptide comprising DNA-directed DNApolymerase activity to form an amplification reaction composition. Whena DNA polymerase having both RNA-directed and DNA-directed polymeraseactivities is used in the reverse transcription reaction above, afurther peptide comprising DNA-directed DNA polymerase does not need tobe added. The amplification reaction composition is thermocycled underconditions suitable to allow amplified products to be generated (shownas “Amplification” in FIG. 1). The sequence of at least part of theamplified product is determined, which allows the corresponding RNAmolecule to be detected (shown as “Sequence Determination” in FIG. 1).

According to one exemplary embodiment, depicted schematically in FIG. 3,a population of small RNA molecules 33 is combined with a first adaptor31 comprising a first oligonucleotide comprising RNA (shown in the openbox) and a second adaptor 32 and Rnl2 ligase to form a ligation reactioncomposition. The ligation reaction composition is incubated underconditions suitable for annealing to occur and a first adaptor and asecond adaptor anneal with a small RNA molecule to form a ligationtemplate comprising the first adaptor annealed to the 5′-end of the RNAmolecule and the second adaptor annealed to the 3′-end of the small RNAmolecule. The ligase will generate a ligated product 34 by ligating thefirst adaptor to the 5′-end of the RNA molecule and the second adaptorto the 3′-end of the RNA molecule at the ligation junctions (shown assolid dots in FIG. 3 and indicated by arrows). Depending on theconcentration of adaptors and RNA molecules in the ligation reactioncomposition, some unannealed first adaptors 31 and/or second adaptors 32may also be present in the ligation reaction composition. Additionally,particularly when the first and/or second adaptors comprise degeneratesequences, undesired annealed byproduct molecules 35 can also form.

The reaction composition comprising the ligated product is combined withan RNA-directed DNA polymerase and under suitable conditions a reversetranscribed product 36 is generated. The reaction composition comprisingthe reverse transcribed product 36 is combined with ribonuclease H andat least some of the ribonucleosides of the ligated product are digestedand an amplification template 38 is generated. Those in the art willappreciate that, at this point, the amplification template comprises, inessence, the cDNA strand of the reverse transcribed product annealedwith the third oligonucleotide of the second adaptor. The amplificationtemplate 38 is combined with a DNA-directed DNA polymerase, a forwardprimer 310 and a reverse primer 311 to form an amplification reactioncomposition. In this illustrative embodiment, the reverse primer furthercomprises an identification sequence 312, sometimes referred to as a“bar code” sequence. If all of the reverse primers in a givenamplification reaction composition comprise the same identificationsequence and that sequence is incorporated into subsequent amplicons,then all of the amplicons generated from the same amplification reactioncomposition can be identified as having come from that reactioncomposition.

The amplification reaction composition is temperature cycled to allowthe polymerase chain reaction to occur and a plurality of amplifiedproducts is generated. In this illustrative embodiment, the amplifiedproducts are purified using polyacrylamide gel electrophoresis (PAGE)and/or high performance liquid chromatography (HPLC; sometimes referredto as high pressure liquid chromatography). The purified amplifiedproducts are sequenced using any technique known in the art, and the RNAmolecule corresponding to that sequence is identified. Those in the artwill appreciate that the disclosed method may be useful for a variety ofanalyses, including without limitation, expression profiling,quantitating one or more specific RNA molecules in one or morecorresponding samples (e.g., with and without drug treatment; amalignant/tumor tissue and the corresponding normal tissue sample;developmental studies using corresponding embryonic, neonatal,adolescent, and/or adult tissues), and small RNA discovery.

It is to be appreciated that if multiple libraries of amplified productare to be generated, each amplified product library can be identified bya unique identification sequence or barcode for that library. In someembodiments, the PCR primer mix for a given amplified product librarycomprises a forward primer (for illustration purposes, see forwardprimer 310 in FIG. 3) and a reverse primer that contains a uniqueidentification sequence or barcode (for illustration purposes, seereverse primer 311 comprising identifier sequence 312 in FIG. 3). Whenan amplification reaction composition comprising such a primer pair iscycled, the barcode is becomes incorporated into the amplified productsof that library. Thus, the exemplary first primer can be matched withany of these exemplary reverse primers in an amplification reactioncomposition to generate a library of amplified products comprising thebarcode of the reverse primer or its complement.

For illustration purposes but not as a limitation, each amplifiedproduct in a first library generated using a PCR primer mix includingthe exemplary forward primer and exemplary reverse primer BC1 (seeExample 11) will contain the barcode sequence AAGCCC and/or itscomplement; while each amplified product in a second library generatedusing a PCR primer mix including the exemplary forward primer andexemplary reverse primer BC2 will contain the barcode sequence CACACCand/or its complement; and so forth. Thus, multiple libraries ofamplified product can be pooled prior to sequencing and the RNAmolecules in the starting material corresponding to each library can beidentified using the target (RNA molecule) sequence or at least part ofthat sequence combined with the barcode or identification sequence forthat library. Those in the art will appreciate that variousidentification sequences can be employed to uniquely mark the amplifiedproducts generated in a given amplification reaction composition.

FIG. 4 schematically depicts another exemplary embodiment of the currentteachings. According to this embodiment, the first and second adaptorsare hybridized with the RNA molecule (shown as Adaptor Hybridization inFIG. 4) and under suitable conditions and in the presence of anappropriate ligase, ligated product is generated (shown as Ligation inFIG. 4). Reverse transcriptase is added to the ligated product and areverse transcribed product is generated under suitable conditions(shown as Reverse Transcription in FIG. 4). The reverse transcribedproduct is digested with ribonuclease H and an amplification template isformed (shown as RNase H in FIG. 4). An amplification reactioncomposition is formed comprising the amplification template, forwardprimer, reverse primer, and a DNA-directed DNA polymerase. Theamplification reaction composition is thermocycled for a number ofcycles that stays within a linear range of amplification (generally,˜12-15 cycles or 12-18 cycles according to one exemplary embodiment),allowing the polymerase chain reaction to occur and amplified product tobe generated (shown as PCR in FIG. 4). In this illustrative embodiment,the amplified product is gel purified (shown as Gel Purif. in FIG. 4)resulting in purified amplified product. Provided that appropriatelysize fractionated or fragmented RNA molecules were used, the amplifiedproduct comprises an insert sequence (shown by a curved bracket in FIG.4; in certain embodiments, insert sizes are about 15 base pairs to about100 base pairs), a first primer region (shown as P1 in FIG. 4), and asecond primer region (shown as P2 in FIG. 4) that includes a bar code oridentification sequence (shown as be in FIG. 4).

As used herein, the terms “polynucleotide”, “oligonucleotide”, and“nucleic acid” are used interchangeably and refer to single-stranded anddouble-stranded polymers of nucleoside monomers, including2′-deoxyribonucleosides (DNA) and ribonucleosides (RNA) linked byinternucleotide phosphodiester bond linkages, or internucleotideanalogs, and associated counter ions, e.g., H⁺, NH₄ ⁺, trialkylammonium,Mg²⁺, Na⁺, and the like. A polynucleotide may be composed entirely ofdeoxyribonucleosides, entirely of ribonucleosides, or chimeric mixturesthereof. As further described below, for example, first adaptors includea first oligonucleotide having at least two ribonucleosides on its 3′end. First oligonucleotides can have 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 20, 25, or more ribonucleosides and in some embodiments, theribonucleosides are contiguous. In some embodiments, second, third orfourth oligonucleotides comprise deoxyribonucleotides. The nucleotidemonomer units may comprise any of the nucleotides described herein,including, but not limited to, nucleotides and nucleotide analogs.Polynucleotides typically range in size from a few monomeric units, e.g.5-40 or 5-60 when they are sometimes referred to in the art asoligonucleotides, to several thousands of monomeric nucleotide units.Unless denoted otherwise, whenever a polynucleotide sequence isrepresented, it will be understood that the nucleotides are in 5′ to 3′order from left to right.

RNA Molecule to be Detected:

In some embodiments, the RNA molecules of the current teachings comprisetotal RNA, a subset or fraction of total RNA, or both. In someembodiments, a sample comprising the RNA molecule to be detectedcomprises all of the RNA obtained from a particular sample or pool ofsamples. In other embodiments, the total RNA is fractionated intosubsets and the RNA molecule to be detected is present in one or more ofthe fractionated subsets. Typically, RNA molecules are extracted from asample using any technique known in the art that yields total RNA or asubset of RNA molecules in the sample.

In some embodiments, the RNA molecules to be detected are fragmented,typically prior to forming the ligation reaction composition. In someembodiments, total RNA can be fragmented or fractionated RNA can befragmented and analyzed using methods provided herein. In someembodiments, the RNA molecule to be detected comprises a plurality ofdifferent RNA species, including without limitation, a plurality ofdifferent mRNA species, which may or may not be fragmented prior togenerating ligation products. In some embodiments the RNA is fragmentedchemically, enzymatically, mechanically, by heating, or combinationsthereof using methods well known in the art. Fragmented RNA is analyzedusing methods provided herein. Whole transcriptome analysis can thus becarried out wherein sequences that are transcribed from DNA areanalyzed, including coding RNA (e.g., for expression analysis) ornoncoding RNA.

In some embodiments, small RNA molecules and/or fragmented RNA in acertain size range are obtained from a sample, for example using a sizefractionation procedure. In some embodiments, the total RNA isfragmented and may also be size fractionated prior to ligating the firstand second adaptors; while in other embodiments total RNA is used in theligation reaction composition. For illustration purposes, but not as alimitation, certain fractionation techniques are depicted in FIG. 11.

In some embodiments, a poly A selection process is performed to separatemessenger RNA (mRNA) from those RNA molecules that lack poly A (poly Aminus RNA molecules). In some embodiments, the total RNA is fractionatedinto subsets by separating at least some of the mRNA from the total RNA,for example but not limited to, using a polyA selection technique knownin the art, including without limitation, oligo-dT chromatography. Insuch embodiments, either the poly A+ fraction or the poly A depletedfraction may be employed in the current teachings to detect at leastsome of the RNA molecules that are present in that fraction. In someembodiments, both fractions may be used separately to detect at leastsome of the RNA molecules that are present in each fraction. In someembodiments, the RNA molecules of interest comprise poly A minus RNAmolecules, for example but not limited to large non-coding RNA.

In certain embodiments, a population of mRNA molecules or a populationof poly A minus RNA molecules is depleted of at least one species ofabundant RNA molecule in the population, for example but not limited to,ribosomal RNA, or mRNA from housekeeping or highly expressed genes,including without limitation, actin mRNA and globin mRNA. For example,certain mRNAs or classes of RNA are depleted from the total RNA, forexample but not limited to, high copy number mRNAs such as actin, GAPDH,globin and other “housekeeping” mRNA; and classes of RNA for example butnot limited to 18S RNA and 28S RNA (for example, using commerciallyavailable kits such as the RiboMinus (Invitrogen, Carlsbad, Calif.) orGLOBINcIear™ (Ambion, Austin, Tex.) Kits (see also U.S. PatentApplication Publication US 2006/0257902, Methods and Compositions forDepleting Abundant RNA Transcripts).

The term chemical fragmentation is used in a broad sense herein andincludes without limitation, exposing the sample comprising the RNA tometal ions, for example but not limited to, zinc (Zn²⁺), magnesium(Mg²⁺), and manganese (Mn²⁺) and heat.

The term enzymatic fragmentation is used in a broad sense and includescombining the sample comprising the RNA with a peptide comprisingnuclease activity, such as an endoribonuclease or an exoribonuclease,under conditions suitable for the peptide to cleave or digest at leastsome of the RNA molecules. Exemplary nucleases include withoutlimitation, ribonucleases (RNases) such as RNase A, RNase T1, RNase T2,RNase U2, RNase PhyM, RNase III, RNase PH, ribonuclease V1,oligoribonuclease (e.g., EC 3.1.13.3), exoribonuclease I (e.g., EC3.1.11.1), and exoribonuclease II (e.g., EC 3.1.13.1), however anypeptide that catalyzes the hydrolysis of an RNA molecule into one ormore smaller constituent components is within the contemplation of thecurrent teachings. Fragmentation of RNA molecules by nucleic acids, forexample but not limited to, ribozymes, is also within the scope of thecurrent teachings.

The term mechanical fragmentation is used in a broad sense and includesany method by which nucleic acids are fragmented upon exposure to amechanical force, including without limitation, sonication, collision orphysical impact, and shear forces.

In some embodiments, very small fragments of RNA are removed using a“clean up” step, for example but not limited to, purification using gelelectrophoresis, glass fiber filters or using magnetic beads, prior tousing the remaining larger RNA molecules according to the currentteachings.

In certain embodiments, the methods of the current teachings employ RNAmolecules that were fractionated using a physical separation method,including without limitation, size separation methods such ascentrifugation, column chromatography/gel sieving, and electrophoreticseparation. In some embodiments, electrophoretic separation of RNAmolecules of interest comprise the flashPAGE™ Fractionator System(Ambion, Austin, Tex.) or size selection by slicing a band from anagarose or polyacrylamide gel according to methods known in the art. Insome embodiments, the RNA molecules used in certain disclosed methodscan be obtained by extracting a subset of RNA molecules in a sampleusing any of a variety of sample preparation kits and reagents,including without limitation, the mirVana™ miRNA Isolation Kit (Ambion).In some embodiments, RNA may be immunoprecipitated.

The terms non-coding RNA or ncRNA refer to any RNA molecule, regardlessof size, that is not translated into a protein. Exemplary ncRNAs includetransfer RNA (tRNA), ribosomal RNA (rRNA), microRNA (miRNA), smallnuclear RNA (snRNA), small nucleolar RNA (snoRNA), guide RNA (gRNA),efference RNA (eRNA), Piwi-interacting RNA (piRNA), Repeat-associatedsiRNA (rasiRNA), signal recognition particle RNA, promoter RNA (pRNA),small interfering RNA (siRNA), and transfer-messenger RNA (tmRNA).

Those of skill in the art will appreciate that the length of RNAmolecule to be detected is not a limitation of the current teachingssince longer molecules can be fractionated and/or fragmented and thefractions and/or fragments detected so as to reconstruct the RNAmolecule. In some embodiments, the length of the RNA molecule to bedetected is 12 to 500 nucleotides, 15 to 110 nucleotides, 15 to 100nucleotides, 18 to 110 nucleotides, 20 to 80 nucleotides, 25 to about 60nucleotides, 20 to about 45 nucleotides, 20 to about 41 nucleotides, 20to about 40 nucleotides, 21, 22, 23, 24, or 25 to about 36, 37, 38, 39,40, or 41 nucleotides, or any integer range therebetween. In someembodiments, the RNA molecule to be detected has a length of 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, or 41 nucleotides.

Those in the art will appreciate that the techniques used to fractionateor fragment RNA is not a limitation of the current teachings and thatvarious fractionation or fragmentation techniques can typically beemployed, depending on which fraction(s) or fragment(s) of RNA moleculesis to be detected.

In certain embodiments, the starting material comprises at least onesynthetic RNA molecule, such as a spike-in control that may be used for,among other things, calibration or standardization. In some embodiments,at least one synthetic RNA molecule species is added to a samplecomprising naturally occurring RNA molecules and the presence of atleast one synthetic RNA species and at least one naturally occurring RNAspecies is detected according to the disclosed methods.

In some embodiments, the RNA molecule to be detected has a5′-monophosphate and a 3′-hydroxyl for efficient ligation. For example,some small RNA biogenesis results in RNA molecules with 5′-endscomprising triphosphates. According to certain embodiments of thecurrent teachings, such RNA molecules are not suitable for adaptorligation and amplification. Thus, intact mRNA molecules with a 5′ capstructure, and RNA molecules with a 5′ triphosphate, including smallRNAs such as endogenous siRNA from C. elegans (Pak 2007), cannot beeffectively ligated to the hybridized adaptors in the reactioncomposition, unless they are first treated with a decapping enzyme, forexample but not limited to, tobacco acid pyrophosphatase (TAP), nucleaseP1, Dcp1p decapping enzyme, Dcp2 decapping enzyme, or DcpS decappingenzyme to convert the 5′ ends of RNA molecules to 5′ monophosphates.Where the RNA molecule of interest comprises 5′-triphosphates, certainembodiments of the current teachings employ tobacco acid pyrophosphataseto convert the 5′-ends of RNA molecules to 5′ monophosphates, renderingthem suitable for ligation.

In some embodiments, fragments generated by certain fragmentationtechniques do not initially possess a terminus that is suitable forenzymatic ligation; in some embodiments, such fragments are treated witha kinase, for example but not limited to, bacteriophage T4polynucleotide kinase, to render the 5′-ends or 3′-ends suitable forligating according to the current teachings.

RNA to be detected may be single stranded or double stranded since theRNA to be detected is combined with at least one first adaptor, at leastone second adaptor, and annealed such that a polypeptide comprisingdouble-strand specific RNA ligase can form a ligated product.

According to the current teachings, an RNA molecule of interest can beeither synthetic or naturally occurring. RNA molecules can besynthesized using oligonucleotide synthesis methods that are well-knownin the art. RNA molecules can also be synthesized biochemically, in vivoor in vitro, according to methods known in the art, for example but notlimited to in vitro transcription techniques, including withoutlimitation, U.S. Pat. Nos. 5,958,688; 5,723,290; 5,514,545; 5,021,335;5,168,038; 5,545,522; 5,716,785; 5,891,636; and 6,291,170. Detaileddescriptions of such techniques can be found in, among other places,Current Protocols in Nucleic Acid Chemistry, Beaucage et al., eds., JohnWiley & Sons, New York, N.Y., including updates through May 2005(hereinafter “Beaucage et al.”); and Blackburn and Gait. Automatednucleic acid synthesizers useful for synthesizing RNA molecules,adaptors, and primers are commercially available from numerous sources,including for example, Applied Biosystems (Foster City, Calif.). RNAmolecules, adaptors, and primers can also be generated biosynthetically,using in vivo methodologies and/or in vitro methodologies that are wellknown in the art. Descriptions of such technologies can be found in,among other places, Sambrook et al. and Ausubel et al. Nucleosideanalogs, such as 2′-OMe-, LNA-, halo-, or arabino-derivatives, forexample, or universal nucleobases can be incorporated into adaptors aslong as the fourth oligonucleotide is a primeable substrate. Purified orpartially purified RNA is commercially available from numerous sources,including FirstChoice® Total RNA, FirstChoice® Poly(A), FirstChoice®Tumor RNA, and the mirVana™ miRNA Reference Panel (Ambion, Austin,Tex.); Reference Total RNA, Human and Mouse, and Universal ReferenceRNAs (Stratagene, La Jolla, Calif.); and the American Type CultureCollection (ATCC), Manassas, Va.

In some embodiments, the RNA molecule to be detected is present in asample. The term “sample” is used in a broad sense herein and isintended to include a wide range of biological materials as well ascompositions derived or extracted from such biological materialscomprising or suspected of comprising RNA. Exemplary samples includewhole blood; red blood cells; white blood cells; buffy coat; hair; nailsand cuticle material; swabs, including buccal swabs, throat swabs,vaginal swabs, urethral swabs, cervical swabs, rectal swabs, lesionswabs, abcess swabs, nasopharyngeal swabs, and the like; urine; sputum;saliva; semen; lymphatic fluid; amniotic fluid; cerebrospinal fluid;peritoneal effusions; pleural effusions; fluid from cysts; synovialfluid; vitreous humor; aqueous humor; bursa fluid; eye washes; eyeaspirates; plasma; pulmonary lavages; lung aspirates; and tissues,including, liver, spleen, kidney, lung, intestine, brain, heart, muscle,pancreas, biopsy material, and the like. The skilled artisan willappreciate that lysates, extracts, or materials obtained from any of theabove exemplary biological samples are also within the scope of thecurrent teachings. Tissue culture cells, including explanted material,primary cells, secondary cell lines, and the like, as well as lysates,extracts, or materials obtained from any cells, are also within themeaning of the term biological sample as used herein. Materialscomprising or suspected of comprising at least one RNA molecule that areobtained from forensic, agricultural, and/or environmental settings arealso within the intended meaning of the term sample. In certainembodiments, a sample comprises a synthetic nucleic acid sequence. Insome embodiments, a sample is totally synthetic, for example but notlimited to, a control sample comprising a buffer solution containing atleast one synthetic nucleic acid sequence. In certain embodiments, thesample is an environmental sample, such as a soil, water, or air sample.

Plant miRNAs can have a 2′-O-methyl group at the 3′ end and can beligated in a ligation reaction as cited herein. However, the efficiencyof such a ligation will be reduced compared to RNA species with a 2′-OHat the 3′ end.

First Adaptors and Second Adaptors:

As stated above, at least one first adaptor comprises a firstoligonucleotide comprising at least two ribonucleosides on the 3′-endand a second oligonucleotide that comprises a single-stranded 5′ portionwhen the first oligonucleotide and the second oligonucleotide arehybridized together as depicted in FIG. 2A. First and secondoligonucleotides are designed to be substantially complementary with theexception of the single stranded portion, further described below. Thesubstantially complementary portion can have a length of 10 to 60nucleotides. In some embodiments, the substantially complementaryportion can have a length of 10 to 40 nucleotides, 12 or 15 to 30nucleotides, 20, 21, 22, or 23 to 25, 27 or 29 nucleotides, or anyinteger range between any of these ranges. When annealed or duplexed toform a first adaptor, the first and second oligonucleotides can have oneblunt end (as in FIG. 2A and FIG. 2B) or can have an overhang of 1, 2,or 3 nucleotides at the end opposite the end having a single strandedportion. The overhang may be on either the first or the secondoligonucleotide.

Also as stated above, at least one second adaptor comprises a thirdoligonucleotide that comprises a 5′ phosphate group and a fourtholigonucleotide that comprises a single-stranded 3′ portion when thethird oligonucleotide and the fourth oligonucleotide are hybridizedtogether also as depicted in FIG. 2A. Third and fourth oligonucleotidesare designed to be substantially complementary with the exception of asingle stranded portion, further described below. The substantiallycomplementary portion can have a length of 10 to 60 nucleotides. Whenannealed or duplexed to form a second adaptor, the third and fourtholigonucleotides can have one blunt end (as in FIG. 2A and FIG. 2B) orcan have an overhang of 1, 2, or 3 nucleotides at the end opposite theend having a single stranded portion. The overhang may be on either thefirst or the second oligonucleotide.

In some embodiments, first, second, third and fourth oligonucleotidesindependently comprise deoxyribonucleosides, ribonucleotides, or bothdeoxyribonucleotides and ribonucleotides with the exception that thefirst oligonucleotide comprises at least two ribonucleosides on the3′-end. It is to be appreciated that in the illustrative embodiment ofFIG. 2A, all of the nucleosides in the first oligonucleotide areribonucleosides, but that in other embodiments as many as all and as fewas two of the nucleosides of the first oligonucleotide can beribonucleosides, provided that the two 3′-most nucleosides of the firstoligonucleotide are ribonucleosides; the remainder of the firstoligonucleotide may comprise ribonucleosides, deoxyribonucleosides, or acombination of both. In some embodiments, the second, third and fourtholigonucleotides comprise deoxyribonucleosides and the firstoligonucleotide comprises all ribonucleosides. In some embodiments, thefirst oligonucleotide comprises ribonucleosides and the second, thirdand fourth oligonucleotides comprise deoxyribonucleotides. The length ofoligonucleotides of first and second adaptors is independent of eachother.

The sequences of the first, second, third and fourth oligonucleotidesare such that substantial complementarity is achieved in the duplexedportion of the adaptors as described above. In some embodiments, thesequences of the first and the third oligonucleotides are different. Thespecific sequence of nucleotides of the duplexed portions of theadaptors is not limiting for the methods herein. In some embodiments, aportion of an adaptor sequence comprises a “promoter sequence,”including without limitation a sequence suitable for initiatingtranscription using a suitable polymerase, for example but not limitedto, T3 RNA polymerase, T7 RNA polymerase, or SP6 RNA polymerase. In someembodiments, a first adaptor comprises a “promoter sequence” for a firstpromoter and a second adaptor comprises a “promoter sequence” for asecond promoter.

The 3′-end of the first oligonucleotide and the 5′-end of the thirdoligonucleotide are suitable for ligation to an RNA molecule to bedetected, which also is suitable for ligation. Oligonucleotides“suitable for ligation” refers to at least one RNA molecule to bedetected, and at least one first adaptor and/or at least one secondadaptor, each comprising an appropriate reactive group. Exemplaryreactive groups include, but are not limited to, a free hydroxyl groupon the 3′ end of the first oligonucleotide of a first adaptor and a freephosphate group on the 5′ end of the RNA molecule to be detected, a freehydroxyl group on the 3′ end of the RNA molecule to be detected and afree phosphate group on the 5′ end of the third oligonucleotide of asecond adaptor.

Single-Stranded Portions of Adaptors:

The single-stranded portions of the illustrative second and fourtholigonucleotides are depicted in FIG. 2A as degenerate hexamer sequences(shown as NNNNNN). However, the use of first and/or second adaptors withsequence-specific single-stranded portions and also longer and shortersingle-stranded portions is within the scope of the current teachings.

In some embodiments, the single-stranded portions comprise,independently, deoxyribonucleosides, ribonucleosides or a combination ofdeoxyribonucleosides and ribonucleosides. In some embodiments, thesingle-stranded portions comprise deoxyribonucleosides.

In some embodiments of single-stranded portions of adaptors, the lengthof the single-stranded portion is as short as one nucleotide and as longas 8 nucleotides. In some embodiments, the length of the single-strandedportion is 2, 4, 6, or 8 nucleotides. In some embodiments, the length ofthe single-stranded portion is 4 or 6 nucleotides. The length of thesingle-stranded portion of the second oligonucleotide is independent ofthe length of the single-stranded portion of the fourth oligonucleotide.

In some embodiments the nucleoside sequence of a single-stranded portionis designed to be complementary to a 5′-sequence or a 3′-sequence of aspecific RNA molecule to be detected. In some embodiments, the specificRNA molecule to be detected hybridizes with the single-stranded portionof at least one first adaptor and the single-stranded portion of atleast one second adaptor such that a ligation reaction can occur. Forhybridizing to specific sequences in some embodiments, the length of thesingle-stranded portions are independently 4 to 6 nucleotides long. Insuch a method, the RNA molecule is directionally detected by methodsherein. One of ordinary skill in the art can design a single-strandedportion corresponding to the 5′-sequence or a 3′-sequence of the RNAmolecule to be detected and using the detection methods provided hereindetect either the sense sequence or the antisense sequence correspondingto the RNA molecule.

In some embodiments, the sequence of a single-stranded portion isdesigned to be a degenerate sequence to allow all RNA molecules of asample having complementary to the degenerate sequence to anneal to thesingle-stranded portion of the adaptor. In some embodiments, degeneratesingle-stranded portions have a length of 1 to 8 nucleotides. In someembodiments, degenerate single-stranded portions have a length of 4, 6,or 8 nucleotides. In some embodiments, the degenerate nucleosidesequences are deoxyribonucleotides.

In some embodiments, the sequence of a single-stranded portion of asecond or fourth oligonucleotide is a degenerate sequence and thesequence of the other of the second or fourth oligonucleotide is asequence corresponding to the RNA molecule to be detected.

Annealing or Hybridizing:

The terms “annealing” and “hybridizing” including, without limitation,variations of the root words hybridize and anneal, are usedinterchangeably and mean the nucleotide base-pairing interaction of onenucleic acid with another nucleic acid that results in the formation ofa duplex, triplex, or other higher-ordered structure. The primaryinteraction is typically nucleotide base specific, e.g., A:T, A:U, andG:C, by Watson-Crick and Hoogsteen-type hydrogen bonding. In certainembodiments, base-stacking and hydrophobic interactions may alsocontribute to duplex stability. For example, conditions under whichprimers anneal to complementary or substantially complementary sequencesare well known in the art, e.g., as described in Nucleic AcidHybridization, A Practical Approach, Hames and Higgins, eds., IRL Press,Washington, D.C. (1985) and Wetmur and Davidson, Mol. Biol. 31:349,1968. In general, whether annealing takes place is influenced by, amongother things, the length of the complementary portion of the nucleicacids, the pH, the temperature, the presence of mono- and divalentcations, the proportion of G and C nucleotides in the hybridizingregion, the viscosity of the medium, and the presence of denaturants.Such variables influence the time required for hybridization. Thepresence of certain nucleotide analogs or minor groove binders in thecomplementary portions of nucleic acids can also influence hybridizationconditions. Thus, the preferred annealing conditions will depend uponthe particular application. Such conditions, however, can be routinelydetermined by persons of ordinary skill in the art, without undueexperimentation. Typically, annealing conditions are selected to allownucleic acids to selectively hybridize with a complementary orsubstantially complementary sequence, but not hybridize to anysignificant degree to other sequences in the reaction.

The term “selectively hybridize” and variations thereof means that,under suitable conditions, a given sequence anneals with a secondsequence comprising a complementary or a substantially complementarystring of nucleotides, but does not anneal to undesired sequences. Inthis application, a statement that one sequence selectively hybridizesor anneals with another sequence encompasses situations where theentirety of both of the sequences hybridize to one another, andsituations where only a portion of one or both of the sequenceshybridizes to the entire other sequence or to a portion of the othersequence. For the purposes of this definition, the term “sequence”includes nucleic acid sequences, polynucleotides, oligonucleotides,primers, target-specific portions, amplification product-specificportions, primer-binding sites, hybridization tags, and hybridizationtag complements.

The term “corresponding” as used herein refers to at least one specificrelationship between the elements to which the term relates. Forexample, a single-stranded 5′ portion of a first adaptor corresponds toa RNA molecule having a terminal nucleotide sequence that hybridizes tothe single-stranded portion. A single-stranded 3′ portion of a secondadaptor corresponds to a RNA molecule having a terminal nucleotidesequence that hybridizes to the single-stranded portion. Furtherexamples include where a primer binds to the corresponding complementaryor substantially complementary primer-binding portion of a nucleic acid,where a particular affinity tag binds to the corresponding affinity tag,for example but not limited to, biotin binding to streptavidin, andwhere a particular hybridization tag anneals with its correspondinghybridization tag complement; and the like.

In this application, a statement that one sequence is the same as,substantially the same as, complementary to, or substantiallycomplementary to another sequence encompasses situations where both ofthe sequences are completely the same as, substantially the same as, orcomplementary or substantially complementary to one another, andsituations where only a portion of one of the sequences is the same as,substantially the same as, complementary to, or substantiallycomplementary to a portion or the entire other sequence. For thepurposes of this definition, the term “sequence” includes RNA, DNA,polynucleotides, oligonucleotides, primers, ligated products, reversetranscribed products, amplification templates, amplified products,primer-binding sites, hybridization tags, and hybridization tagcomplements.

The terms “denaturing” or “denaturation” as used herein refer to anyprocess in which a double-stranded polynucleotide, including adouble-stranded amplification product or a double-stranded DNA or aDNA:RNA duplex is converted to two single-stranded polynucleotides.Denaturing a double-stranded polynucleotide includes without limitation,a variety of thermal or chemical techniques for denaturing a duplex,thereby releasing its two single-stranded components. Those in the artwill appreciate that the denaturing technique employed is generally notlimiting unless it inhibits or appreciably interferes with a subsequentamplifying and/or detection step.

Ligation:

The term “ligating,” or forms thereof, is used herein to refer to anenzymatic ligation process that uses a polypeptide comprisingdouble-strand specific RNA ligase activity in which an inter-nucleotidelinkage is formed between immediately adjacent ends of oligonucleotidesthat are adjacently hybridized to a template. Formation of the linkageis double-strand dependent and specific, also termed duplex-dependentand specific or template-dependent and specific. The internucleotidelinkage can include, but is not limited to, phosphodiester bondformation between a 3′-ribonucleoside and a 5′-ribonucleotide, or a3′-ribonucleoside and a 5′-deoxyribonucleoside. The term “double-strandspecific RNA ligase” as used herein refers to a polypeptide comprisingRNA ligase activity that preferentially seals or ligates a nick betweenan oligonucleotide having a 3′-terminal ribonucleotide and anoligonucleotide having a 5′ phosphate group, specifically when theoligonucleotides are immediately adjacently hybridized to a templatemolecule. For example, but without limitation, a nick between the 3′-endof the first oligonucleotide of the first adaptor and the RNA moleculeto which the first adaptor is annealed is schematically presented asligation junction 24 in FIG. 2B.

In certain embodiments, the polypeptide comprising double-strandspecific RNA ligase activity is an Rnl2 family ligase exemplified by thebacteriophage T4 RNA ligase 2 (T4 Rnl2), including without limitation,an enzymatically active mutant or variant of Rnl2. T4 Rnl2 is aprototype ligase for an RNA ligase family that differs from the Rnl1family of ligases due to variant nucleotidyl transferase motifs (see,e.g., Ho and Shuman, Proc. Natl. Acad. Sci. 99(20):12709-14 (2002); andYin et al., J. Biol. Chem. 278:17601-08 (2003)). The T4 Rnl2 familyincludes vibriophage KVP40 Rnl2, the RNA-editing ligases (RELs) ofTrypanosoma brucei (TbREL1 and TbREL2) and of Leishmania tarentolae(LtREL1 and LtREL2), poxvirus AmEPV (entomopoxvirus) ligase, baculovirusAcNPV ligase, and baculovirus XcGV ligase, among others. In someembodiments using REL ligases, the second and fourth oligonucleotidescan comprise ribonucleotides and, in certain embodiments, the singlestranded portions of the second and fourth oligonucleotides can compriseribonucleotides.

T4 Rnl2 ligase is commercially available from NEW ENGLAND BIOLABS®(Ipswich, Mass.) or the ligase can be isolated as described inNandakumar et al., JBC 280(25):23484-23489, 2005; Nandakumar et al., JBC279(30):31337-31347, 2004; and Nandakumar et al., Molecular Cell16:211-221, 2004. The T4 Rnl2 enzyme is encoded by gene gp24.1 of phageT4. In certain embodiments, a polypeptide comprising ligase activitycomprises T4 Rnl2 or another member of the Rnl2 family of ligases, or anenzymatically active mutant or variant thereof.

In certain embodiments, the polypeptide comprising double-strandspecific RNA ligase activity is a Deinococcus radiodurans RNA ligase(DraRnl) (Raymond et al., Nucl Acids Res 35(3):839-849, 2007), or aDraRnl-type ligase, including without limitation, a ligase having GenBank accession no. XP_(—)367846 from the fungi Magnaporthe grisea, GenBank accession no. CAE76396 from Neurospora crassa, accession no.XP_(—)380758 from Gibberella zeae, or Accession no. EAL61744 from theamoeba Dictyostelium discoideum. In some embodiments, a ligase caninclude a combination of any of the above-cited ligases, orenzymatically active mutants or variants thereof.

In certain embodiments, a polypeptide comprising double-strand specificRNA ligase activity can be preadenylated, the 5′-terminal nucleotide ofthe third oligonucleotide can be preadenylated, or the 5′-terminalnucleotide of the RNA molecule to be detected can be preadenylated, or acombination thereof. Ho et al. (Structure 12:327-339) sets forth amechanism for T4 Rnl2 where the C-terminal domain thereof functions insealing 3′-OH and 5′-P RNA ends. The N-terminal segment (1-249) of theRnl2 protein is reported to function as an autonomousadenylyltransferase/App-RNA ligase domain. In general, RNA ligases join3′-OH and 5′-PO₄ RNA termini through a series of three nucleotidyltransfer steps involving activated covalent intermediates. RNA ligasereacts with ATP to form a covalent ligase-AMP intermediate pluspyrophosphate. AMP is then transferred from ligase-adenylate to a 5′-PO₄RNA end to form an RNA-adenylate intermediate (AppRNA). Ligase thencatalyzes attack by an RNA 3′-OH on the RNA-adenylate to seal the twoends via a phosphodiester bond and release AMP. Mechanisms for RNAligation are further discussed by Nandakumar et al. (ibid 2005, 2004a,2004b) Yin et al. (JBC 278:20, 17601-17608; Virology 319:141-151, 2004),Ho et al. (ibid; PNAS, 99:20, 12709-12714, 2002), Gumport et al. (inGene Amplification and Analysis, Vol 2, edited by Chirikjian, J. G., andPapas, T. S., 1981, 313-345) and by Raymond et al. (Nucleic Acids Res.35:3, 839-849, 2007). Preadenylated agents such as ligase-adenylate,RNA-adenylate, or a chimeric DNA/RNA-adenylate are contemplated for usein some embodiments of the current teachings.

According to certain embodiments, at least one species of first adaptor,at least one species of second adaptor, at least one species of RNAmolecule, and a polypeptide comprising double-strand specific RNA ligaseactivity are combined in a ligation reaction composition. It is to beappreciated that the adaptors of the current teachings are each ligatedwith the corresponding RNA molecule in the same reaction compositionduring the same incubation period, that is simultaneously or nearlysimultaneously, in contrast to other techniques in which one adaptor isligated to one end of an RNA molecule of interest in one reaction, thenanother adaptor is ligated or incorporated onto the opposite side of thesame RNA molecule of interest in a second reaction, often with anintervening gel purification, phosphorylation, or reverse transcriptionstep (see, e.g., Elbashir et al., Genes and Development 15:188-200,2001; Ambros and Lee, Methods in Mol. Biol. 265:131-58, 2004; Berezikovet al., Nature Genet. Supp. 38:S2-S7, 2006; Takada et al., Nucl. AcidsRes. 34(17):e115, 2006; Michael, Methods in Mol. Biol. 342:189-207,2006; and Takada and Mano, Nature Protocols 2(12):3136-45, 2007). It isto be understood that, with respect to the current teachings, the orderof adding components to the ligation reaction composition is generallynot significant and is intended to be encompassed within the term“forming a ligation reaction composition” or similar terms used herein,unless expressly stated otherwise. Thus, the sequential addition of oneadaptor (for example, a first adaptor) to a reaction compositioncomprising RNA molecules and a ligase, followed by the subsequentaddition of the other adaptor (in this example, the second adaptor) tothat reaction composition is within the intended scope of the instantteachings, regardless of whether there is an incubation step between theaddition of one adaptor and the addition of the other adaptor.

Reverse Transcription:

In some embodiments, detecting the RNA molecule comprises reversetranscribing the ligated product to form a reverse transcribed product.The terms “reverse transcribing” and “reverse transcription” and formsthereof as used herein refer to the process of generating adouble-stranded RNA-DNA hybrid molecule, starting with the ligatedproduct, based on the sequential catalytic addition ofdeoxyribonucleotides or analogs of deoxyribonucleotides to the hybridmolecule in a template dependent manner using a polypeptide havingRNA-directed DNA polymerase transcription activity. According to thecurrent teachings, the fourth oligonucleotide of the second adaptor canserve as the primer for reverse transcribing the ligation product togenerate the reverse transcribed product. Addition of a separate primerfor reverse transcription, therefore, is not necessary.

In some embodiments, the polypeptide having RNA-directed DNA polymerasetranscription activity comprises MMLV reverse transcriptase, includingenzymatically active mutants or variants thereof, for example but notlimited to, ArrayScript™ reverse transcriptase (Ambion), SuperScript™reverse transcriptase (Invitrogen); or a polypeptide comprising reversetranscription activity but that has decreased RNAse H activity whencompared to the corresponding wild-type reverse transcriptase, forexample but not limited to, an “RNase H minus” mutant, such as RNaseH-minus HIV-1 reverse transcriptase (Wu et al., J. Virol.73(6):4794-4805, 1999).

In some embodiments, the RNA-directed DNA polymerase transcriptionactivity can be carried out by a DNA-directed DNA polymerase thatpossesses RNA-directed DNA polymerase activity under certain reactionconditions, for example but not limited to, Tth DNA polymerase and DNApolymerase I from Carboxydothermus hydrogenoformans.

Ribonuclease H Digestion:

In some embodiments, the reverse transcribed product is digested withribonuclease H to remove at least some of the ribonucleosides to form anamplification template. The term “digesting”, particularly in referenceto a ribonuclease, refers to the catalysis of RNA into smallercomponents, for example but not limited to, cleavage of the RNA strandof the reverse transcribed product by RNase H to generate asingle-stranded or substantially single-stranded cDNA molecule that canserve as an amplification template of the current teachings.

Amplification:

The terms “amplifying” and “amplification” are used in a broad sense andrefer to any technique by which at least a part of an amplificationtemplate, at least part of an amplified product, or both, is reproducedor copied (including the synthesis of a complementary copy), typicallyin a template-dependent manner, including without limitation, a broadrange of techniques for amplifying nucleic acid sequences, eitherlinearly or exponentially. Some non-limiting examples of amplificationtechniques include the polymerase chain reaction (PCR) including withoutlimitation, reverse transcription PCR (RT-PCR), asynchronous primer PCR,emulsion PCR (ePCR), quantitative PCR (qPCR), and asymmetric PCR, primerextension, strand displacement amplification (SDA), multipledisplacement amplification (MDA), nucleic acid strand-basedamplification (NASBA), rolling circle amplification (RCA),transcription-mediated amplification (TMA), transcription, and the like,including multiplex versions or combinations thereof. Descriptions ofsuch techniques can be found in, among other places, Sambrook andRussell ibid.; Sambrook et al.; Ausubel et al.; PCR Primer: A LaboratoryManual, Diffenbach, Ed., Cold Spring Harbor Press (1995); The ElectronicProtocol Book, Chang Bioscience (2002); Msuih et al., J. Clin. Micro.34:501-07 (1996); McPherson and Moller, PCR The Basics, Bios ScientificPublishers, Oxford, U.K., 2000 (“McPherson”); Rapley, The Nucleic AcidProtocols Handbook (2000), Humana Press, Totowa, N.J. (“Rapley”); U.S.Pat. Nos. 6,027,998 and 6,511,810; PCT Publication Nos. WO 97/31256 andWO 01/92579; Ehrlich et al., Science 252:1643-50 (1991); Innis et al.,PCR Protocols: A Guide to Methods and Applications, Academic Press(1990); Favis et al., Nature Biotechnology 18:561-64 (2000); Williams etal., Nature Methods 3(7):545-50 (2006); and Rabenau et al., Infection28:97-102 (2000).

Amplification can comprise thermocycling (sometimes referred to ascycling or thermal cycling) or can be performed isothermally. In certainembodiments, amplifying comprises at least one cycle, and typicallymultiple cycles, of the sequential steps of: hybridizing a primer with acomplementary or substantially complementary sequence of anamplification template, an amplified product, or the complement ofeither; synthesizing a strand of nucleotides in a template-dependentmanner using a polymerase; and denaturing the newly-formed nucleic acidduplex to separate the strands. The cycle may or may not be repeated, asdesired. In some embodiments, amplifying comprises a cycling theamplification reaction composition in a thermocycler, for example butnot limited to a GeneAmp® PCR System 9700, 9600, 2700, or 2400thermocycler (all from Applied Biosystems). In certain embodiments,newly-formed nucleic acid duplexes are not initially denatured, but areused in their double-stranded form in one or more subsequent steps andeither or both strands can, but need not, serve as a surrogate for thecorresponding RNA molecule of interest. In certain embodiments,single-stranded amplicons are generated, for example but not limited toasymmetric PCR, asynchronous PCR, or transcription.

Primer extension is an amplifying technique that comprises elongating aprimer that is annealed to a template in the 5′=>3′ direction using anextending enzyme such as a polymerase to form an extension product, forexample but not limited to reverse transcribing a ligated product oramplifying an amplification template or an amplified product. Accordingto certain embodiments, with appropriate buffers, salts, pH,temperature, and nucleotide triphosphates, a polymerase incorporatesnucleotides complementary to the template strand starting at the 3′-endof an annealed primer, to generate a complementary strand. In certainembodiments, the polymerase used for primer extension lacks orsubstantially lacks 5′-exonuclease activity.

In some embodiments, the amplification template is combined with atleast one forward primer, at least one reverse primer, and a polypeptidehaving DNA-directed DNA polymerase activity to form an amplificationreaction composition.

The term “DNA polymerase” is used in a broad sense herein and refers toany polypeptide that is able to catalyze the addition ofdeoxyribonucleotides or analogs of deoxyribonucleotides to a nucleicacid polymer in a template dependent manner for example, but not limitedto, the sequential addition of deoxyribonucleotides to the 3′-end of aprimer that is annealed to a nucleic acid template during a primerextension reaction. Typically DNA polymerases include DNA-directed DNApolymerases and RNA-directed DNA polymerases, including reversetranscriptases. Some reverse transcriptases possess DNA-directed DNApolymerase activity under certain reaction conditions, including AMVreverse transcriptase and MMLV reverse transcriptase. Some DNA-directedDNA polymerases possess reverse transcriptase under certain reactionconditions, for example, but not limited to Thermus thermophilus (Tth)DNA polymerase. Descriptions of DNA polymerases can be found in, amongother places, Lehninger Principles of Biochemistry, 3d ed., Nelson andCox, Worth Publishing, New York, N.Y., 2000, particularly Chapters 26and 29; Twyman, Advanced Molecular Biology: A Concise Reference, BiosScientific Publishers, New York, N.Y., 1999; Ausubel et al., CurrentProtocols in Molecular Biology, John Wiley & Sons, Inc., includingsupplements through May 2005 (hereinafter “Ausubel et al.”); Lin andJaysena, J. Mol. Biol. 271:100-11, 1997; Pavlov et al., Trends inBiotechnol. 22:253-60, 2004; and Enzymatic Resource Guide: Polymerases,1998, Promega, Madison, Wis. Expressly within the intended scope of theterms DNA-directed DNA polymerase and RNA-directed DNA polymerase areenzymatically active mutants or variants thereof, including enzymesmodified to confer different temperature-sensitive properties (see,e.g., U.S. Pat. Nos. 5,773,258; 5,677,152; and 6,183,998; and DNAAmplification: Current Techniques and Applications, Demidov and Broude,eds., Horizon Bioscience, 2004, particularly in Chapter 1.1).

Enzymatically Active Mutants or Variants of Enzymes:

For the purposes of the current teachings, when a specific enzyme or apolypeptide comprising enzymatic activity is described or claimed,enzymatically active mutants or variants of that enzyme/polypeptide areintended to be included, unless specifically stated otherwise. Forillustration purposes but not as a limitation, when the terms “Rnl2” or“Rnl2 ligase” are used in this specification or the appended claims, thenaturally-occurring or wild-type Rnl2 ligase as well as allenzymatically active mutants or variants of Rnl2 ligase are intended tobe included, unless specifically stated otherwise. Similarly, anRNA-directed DNA polymerase, a ribonuclease, or a DNA-directed DNApolymerase, is considered an equivalent to an enzymatically activemutant or variant thereof. The term “enzymatically active mutant orvariant thereof,” refers to one or more polypeptides derived from thecorresponding enzyme that retains at least some of the desired enzymaticactivity, such as ligating, reverse transcribing, digesting, amplifying,or as appropriate. Also within the scope of this term are: enzymaticallyactive fragments, including but not limited to, cleavage products, forexample but not limited to Klenow fragment, Stoffel fragment, orrecombinantly expressed fragments and/or polypeptides that are smallerin size than the corresponding enzyme; mutant forms of the correspondingenzyme, including but not limited to, naturally-occurring mutants, suchas those that vary from the “wild-type” or consensus amino acidsequence, mutants that are generated using physical and/or chemicalmutagens, and genetically engineered mutants, for example but notlimited to random and site-directed mutagenesis techniques; amino acidinsertions and deletions, truncated forms, and changes due to nucleicacid nonsense mutations, missense mutations, and frameshift mutations(see, e.g., Sriskanda and Shuman, Nucl. Acids Res. 26(2):525-31, 1998;Odell et al., Nucl. Acids Res. 31(17):5090-5100, 2003); reversiblymodified nucleases, ligases, and polymerases, for example but notlimited to those described in U.S. Pat. No. 5,773,258; biologicallyactive polypeptides obtained from gene shuffling techniques (see, e.g.,U.S. Pat. Nos. 6,319,714 and 6,159,688), splice variants, both naturallyoccurring and genetically engineered, provided that they are derived, atleast in part, from one or more corresponding enzymes; polypeptidescorresponding at least in part to one or more such enzymes that comprisemodifications to one or more amino acids of the native sequence,including without limitation, adding, removing or alteringglycosylation, disulfide bonds, hydroxyl side chains, and phosphate sidechains, or crosslinking, provided such modified polypeptides retain atleast some of the desired catalytic activity; and the like. Expresslywithin the meaning of the term “enzymatically active mutants or variantsthereof” when used in reference to a particular enzyme(s) areenzymatically active mutants of that enzyme, enzymatically activevariants of that enzyme, or enzymatically active mutants of that enzymeand enzymatically active variants of that enzyme.

The skilled artisan will readily be able to measure enzymatic activityusing an appropriate assay known in the art. Thus, an appropriate assayfor polymerase catalytic activity might include, for example, measuringthe ability of a variant to incorporate, under appropriate conditions,rNTPs or dNTPs into a nascent polynucleotide strand in atemplate-dependent manner. Likewise, an appropriate assay for ligasecatalytic activity might include, for example, the ability to ligateadjacently hybridized oligonucleotides comprising appropriate reactivegroups, such as disclosed herein. Protocols for such assays may befound, among other places, in Sambrook et al., Molecular Cloning, ALaboratory Manual, Cold Spring Harbor Press (1989) (hereinafter“Sambrook et al.”), Sambrook and Russell, editors, Molecular Cloning,Vol 3, 3rd edition, Cold Spring Harbor Press (2001), Ausubel et al., andHousby and Southern, Nucl. Acids Res. 26:4259-66, 1998) and thereferences cited below for the family of Rnl2 ligases.

Amplification Primers:

The term “primer” refers to a polynucleotide that selectively hybridizesto a corresponding primer-binding site of an amplification template, anamplified product, or both; and allows the synthesis of a sequencecomplementary to the corresponding polynucleotide template from its 3′end. A “primer pair” comprises a forward primer and a reverse primerthat anneal to one strand of an amplification product or its complement.Primer pairs are particularly useful in certain exponentialamplification techniques, such as the polymerase chain reaction. Incertain embodiments, a forward primer and the corresponding reverseprimer of a primer pair have different melting temperatures (Tm) topermit asynchronous primer PCR.

As used herein, “forward” and “reverse” are used to indicate relativeorientation of primers on a polynucleotide sequence such as anamplification template or an amplified product. For illustrationpurposes but not as a limitation, consider a single-strandedpolynucleotide drawn in a horizontal, left to right orientation with its5′-end on the left. The “reverse” primer is designed to anneal with thedownstream primer-binding site at or near the “3′-end” of thisillustrative polynucleotide in a 5′ to 3′ orientation, right to left.The corresponding “forward” primer is designed to anneal with thecomplement of the upstream primer-binding site at or near the “5′-end”of the polynucleotide in a 5′ to 3′ “forward” orientation, left toright. Thus, the reverse primer comprises a sequence that iscomplementary to the “reverse” or downstream primer-binding site of thepolynucleotide and the forward primer comprises a sequence that is thesame as the forward or upstream primer-binding site. It is to beunderstood that the terms “3-end” and “5′-end” as used in this paragraphare illustrative only and do not necessarily refer literally to therespective ends of the polynucleotide, as such primer-binding sites maybe located internally. Rather, the only limitation is that the reverseprimer of this exemplary primer pair anneals with a reverseprimer-binding site that is downstream or to the right of the forwardprimer-binding site that comprises the same sequence as thecorresponding forward primer. As will be recognized by those of skill inthe art, these terms are not intended to be limiting, but rather toprovide illustrative orientation in a given embodiment.

A primer may comprise a nucleotide sequence of an adaptoroligonucleotide or a nucleotide sequence corresponding to an adaptoroligonucleotide. For example, forward primer having SEQ ID NO:5comprises the sequence of first oligonucleotide having SEQ ID NO:1 (withT's instead of U's). Some embodiments of relationships between primersand adaptor sequences can be understood by the schematic of FIG. 15 inwhich arrows depict variously, forward and reverse PCR primers orforward and reverse SYBR primers. P1 and P2 refer to primer portions.

As used herein, the term “primer-binding site” refers to a region of apolynucleotide sequence that can serve directly, or by virtue of itscomplement, as the template upon which a primer can anneal for any of avariety of primer nucleotide extension reactions known in the art (forexample, PCR). It will be appreciated by those of skill in the art thatwhen two primer-binding sites are present on a single polynucleotide(for example but not limited to a first extension product or a secondextension product), the orientation of the two primer-binding sites isgenerally different. For example, one primer of a primer pair iscomplementary to and can hybridize with to the first primer-bindingsite, while the corresponding primer of the primer pair is designed tohybridize with the complement of the second primer-binding site. Statedanother way, in some embodiments the first primer-binding site can be ina sense orientation, and the second primer-binding site can be in anantisense orientation. In addition, “universal” primers andprimer-binding sites as used herein are generally chosen to be as uniqueas possible given the particular assays and host genomes to ensurespecificity of the assay.

In some embodiments, a primer and/or an amplified product comprises a“promoter sequence”, including without limitation a sequence suitablefor initiating transcription using a suitable polymerase, for examplebut not limited to, T3 RNA polymerase, T7 RNA polymerase, or SP6 RNApolymerase. Some embodiments of the current teachings employ a“promoter-primer” in a method of incorporating a promoter sequence intoan amplification product. In some embodiments, a promoter sequencecomprises a multiplicity of different sequences suitable for binding anRNA polymerase, for example but not limited to a first sequence suitablefor binding a first RNA polymerase and a second sequence suitable forbinding a second RNA polymerase. Those in the art understand that as anamplification product comprising a promoter sequence is amplified bycertain amplification methods, the complement of the promoter sequencemay be synthesized in the complementary amplicon. Thus, it is to beunderstood that the complement of a promoter sequence is expresslyincluded within the intended meaning of the term promoter sequence, asused herein. Some embodiments of the disclosed methods and kits employ a“promoter-primer” in methods of incorporating a desired promotersequence into an amplification product.

Those in the art understand that as an amplified product is amplified bycertain amplification methods, the complement of the primer-binding siteis synthesized in the complementary amplicon. Thus, it is to beunderstood that the complement of a primer-binding site is expresslyincluded within the intended meaning of the term primer-binding site, asused herein.

In some embodiments, the amplification methods of the current teachingscomprise a Q-PCR reaction. The terms “quantitative PCR”, “real timePCR”, or “Q-PCR” refer to a variety of methods used to quantify theresults of the polymerase chain reaction for specific nucleic acidsequences. Such methods typically are categorized as kinetics-basedsystems that generally determine or compare the amplification factor,such as determining the threshold cycle (CT), or as co-amplificationmethods, that generally compare the amount of product generated fromsimultaneous amplification of target and standard templates. Many Q-PCRtechniques comprise reporter probes, intercalating agents, or both. Forexample but not limited to TaqMan® probes (Applied Biosystems),i-probes, molecular beacons, Eclipse probes, scorpion primers, Lux™primers, FRET primers, ethidium bromide, SYBR® Green I (MolecularProbes), and PicoGreen® (Molecular Probes). In some embodiments,detecting comprises a real-time detection instrument. Exemplaryreal-time instruments include, the ABI PRISM® 7000 Sequence DetectionSystem, the ABI PRISM® 7700 Sequence Detection System, the AppliedBiosystems 7300 Real-Time PCR System, the Applied Biosystems 7500Real-Time PCR System, the Applied Biosystems 7900 HT Fast Real-Time PCRSystem (all from Applied Biosystems); the LightCycler™ System (RocheMolecular); the Mx3000P™ Real-Time PCR System, the Mx3005P™ Real-TimePCR System, and the Mx4000® Multiplex Quantitative PCR System(Stratagene, La Jolla, Calif.); and the Smart Cycler System (Cepheid,distributed by Fisher Scientific). Descriptions of real-time instrumentscan be found in, among other places, their respective manufacturer'susers manuals; McPherson; DNA Amplification: Current Technologies andApplications, Demidov and Broude, eds., Horizon Bioscience, 2004; andU.S. Pat. No. 6,814,934.

In certain embodiments, an amplification reaction comprises multiplexamplification, in which a multiplicity of different amplificationtemplates, a multiplicity of different amplification product species, orboth, are simultaneously amplified using a multiplicity of differentprimer pairs (see, e.g., Henegariu et al., BioTechniques 23:504-11,1997; and Rapley, particularly in Chapter 79). Certain embodiments ofthe disclosed methods comprise a single-plex amplification reaction,including without limitation, an amplification reaction comprising amultiplicity of single-plex amplifications performed in parallel, forexample but not limited to certain TaqMan® Array configurations whereinapproximately 100 nuclease assays are performed in parallel to determinewhether specific amplified products are present and in what quantity.

In certain embodiments, an amplifying reaction comprises asymmetric PCR.According to certain embodiments, asymmetric PCR comprises anamplification composition comprising (i) at least one primer pair inwhich there is an excess of one primer, relative to the correspondingprimer of the primer pair, for example but not limited to a five-fold, aten-fold, or a twenty-fold excess; (ii) at least one primer pair thatcomprises only a forward primer or only a reverse primer; (iii) at leastone primer pair that, during given amplification conditions, comprises aprimer that results in amplification of one strand and a correspondingprimer that is disabled; or (iv) at least one primer pair that meets thedescription of both (i) and (iii) above. Consequently, when anamplification template or an amplification product is amplified, anexcess of one strand of the subsequent amplification product (relativeto its complement) is generated. Descriptions of asymmetric PCR, can befound in, among other places, McPherson, particularly in Chapter 5; andRapley, particularly in Chapter 64.

In certain embodiments, one may use at least one primer pair wherein themelting temperature (Tm₅₀) of one of the primers is higher than the Tm₅₀of the other primer, sometimes referred to as asynchronous primer PCR(A-PCR, see, e.g., U.S. Pat. No. 6,887,664). In certain embodiments, theTm₅₀ of the forward primer is at least 4-15° C. different from the Tm₅₀of the corresponding reverse primer. In certain embodiments, the Tm₅₀ ofthe forward primer is at least 8-15° C. different from the Tm₅₀ of thecorresponding reverse primer. In certain embodiments, the Tm₅₀ of theforward primer is at least 10-15° C. different from the Tm₅₀ of thecorresponding reverse primer. In certain embodiments, the Tm₅₀ of theforward primer is at least 10-12° C. different from the Tm₅₀ of thecorresponding reverse primer. In certain embodiments, in at least oneprimer pair, the Tm₅₀ of a forward primer differs from the Tm₅₀ of thecorresponding reverse primer by at least about 4° C., by at least about8° C., by at least about 10° C., or by at least about 12° C.

In certain amplifying embodiments, in addition to the difference in Tm₅₀of the primers in a primer pair, there is also an excess of one primerrelative to the other primer in the primer pair. In certain embodiments,there is a five- to twenty-fold excess of one primer relative to theother primer in the primer pair. In certain embodiments of A-PCR, theprimer concentration is at least 50 nM.

In A-PCR according to certain embodiments, one may use conventional PCRin the first cycles of amplification such that both primers anneal andboth strands of a double-stranded amplicon are amplified. By raising thetemperature in subsequent cycles of the same amplification reaction,however, one may disable the primer with the lower T_(m) such that onlyone strand is amplified. Thus, the subsequent cycles of A-PCR in whichthe primer with the lower T_(m) is disabled result in asymmetricamplification. Consequently, when the target region or an amplificationproduct is amplified, an excess of one strand of the subsequentamplification product (relative to its complement) is generated.

According to certain embodiments of A-PCR, the level of amplificationcan be controlled by changing the number of cycles during the firstphase of conventional PCR cycling. In such embodiments, by changing thenumber of initial conventional cycles, one may vary the amount of thedouble-stranded amplification products that are subjected to thesubsequent cycles of PCR at the higher temperature in which the primerwith the lower T_(m) is disabled.

In certain embodiments, amplifying comprises in vitro transcription. Insome embodiments, a first adaptor, a second adaptor, a first primer, asecond primer, or combinations thereof, comprise a promoter sequence orits complement, for example but not limited to, a promoter-primer. Insome embodiments, a reverse transcribed product comprising a promoter oran amplified product comprising a promoter is combined withribonucleotide triphosphates, an appropriate buffer system, and asuitable RNA polymerase, for example but not limited to, SP6, T3, or T7RNA polymerase and amplified RNA (aRNA) are generated according to knownmethods. The aRNA may be used for array analysis, such as microarray orbead array analysis, wherein the sequence and quantity of the aRNAspecies can be determined. Thus, in certain embodiments, such aRNAserves as a surrogate for the corresponding RNA molecule.

Certain methods of optimizing amplification reactions are known to thoseskilled in the art. For example, it is known that PCR may be optimizedby altering times and temperatures for annealing, polymerization, anddenaturing, as well as changing the buffers, salts, and other reagentsin the reaction composition. Optimization may also be affected by thedesign of the primers used. For example, the length of the primers, aswell as the G-C:A-T ratio may alter the efficiency of primer annealing,thus altering the amplification reaction. Descriptions of amplificationoptimization can be found in, among other places, James G. Wetmur,“Nucleic Acid Hybrids, Formation and Structure,” in Molecular Biologyand Biotechnology, pp. 605-8, (Robert A. Meyers ed., 1995); McPherson,particularly in Chapter 4; Rapley; and Protocols & Applications Guide,rev. 9/04, Promega Corp., Madison, Wis.

Purifying the amplified product according to the present teachingscomprises any process that removes at least some unligated adaptors,unligated RNA molecules, byproducts, primers, enzymes or othercomponents of the ligation reaction composition, the amplificationreaction composition, or both following at least one cycle ofamplification. Such processes include, but are not limited to, molecularweight/size exclusion processes, e.g., gel filtration chromatography ordialysis, sequence-specific hybridization-based pullout methods,affinity capture techniques, precipitation, adsorption, gelelectrophoresis, conventional cloning, conventional cloning withconcatamerization, or other nucleic acid purification techniques. Insome embodiments purifying the amplified product comprises gelelectrophoresis, including without limitation, polyacrylamide gelelectrophoresis (PAGE) and/or agarose gel electrophoresis. In certainembodiments, the amplified product is purified using high-performanceliquid chromatography (HPLC; sometimes also referred to as high-pressureliquid chromatography).

Detection:

The RNA molecule of interest is detected by detecting the ligatedproduct or a surrogate thereof. In some embodiments, the ligated productis reverse transcribed as described above and the reverse transcribedproduct is placed on an array and detected using standard methods knownby one of skill in the art. In some embodiments, the reverse transcribedproduct is labeled with biotin and detection is by using streptavidinbinding thereto. In some embodiments, the reverse transcribed product ispurified using glass fiber filters, beads or is gel-purified. In someembodiments, the reverse transcribed product is combined with a peptidecomprising ribonuclease activity to form a digestion reactioncomposition and incubated under conditions suitable for digesting atleast some of the ribonucleosides from the reverse transcribed productto form an amplification template.

The terms “detecting” and “detection” are used in a broad sense hereinand encompass any technique by which one can determine whether or not aparticular RNA molecule i.e., an RNA molecule of interest, is present ina sample. In some embodiments, the presence of a surrogate is detected,directly or indirectly, allowing the presence or absence of thecorresponding RNA molecule to be determined. For example, the presenceof a surrogate is detected by detecting a family of labeled sequencingproducts obtained using an amplified product or a ligated product as thetemplate; or detecting the fluorescence generated when a nucleasereporter probe, annealed to an amplified product, is cleaved by apolymerase, wherein the detectable signal or detectable change in signalindicates that the corresponding amplified product and/or ligatedproduct has been amplified and thus the corresponding RNA molecule ispresent in the sample. In some embodiments, detecting comprisesquantitating the detectable signal, including without limitation, areal-time detection method, such as quantitative PCR (“O-PCR”). In someembodiments, detecting comprises determining the sequence of asequencing product or a family of sequencing products generated using anamplification product as the template; in some embodiments, suchdetecting comprises obtaining the sequence of a family of sequencingproducts. In some embodiments, detecting an RNA molecule comprises anucleic acid dye, for example but not limited to, in a Q-PCR reactioncomposition. Those in the art will understand that the ligated products,reverse transcribed products, amplification templates, and amplifiedsequences each serve as a surrogate for the RNA molecule from which theywere directly or indirectly generated and that by detecting any of theseproducts one is directly or indirectly detecting the corresponding RNAmolecule.

The term “reporter probe” refers to a sequence of nucleotides,nucleotide analogs, or nucleotides and nucleotide analogs, thatspecifically anneals with a corresponding amplicon, for example but notlimited to a PCR product, and when detected, including but not limitedto a change in intensity or of emitted wavelength, is used to identify,detect, and/or quantify the corresponding amplicon and thus thecorresponding RNA molecule. Thus, by indirectly detecting the amplicon,one can determine that the corresponding RNA molecule is present in thesample. Most reporter probes can be categorized based on their mode ofaction, for example but not limited to: nuclease probes, includingwithout limitation TaqMan® probes; extension probes including withoutlimitation scorpion primers, Lux™ primers, Amplifluors, and the like;and hybridization probes including without limitation molecular beacons,Eclipse® probes, light-up probes, pairs of singly-labeled reporterprobes, hybridization probe pairs, and the like. In certain embodiments,reporter probes comprise an amide bond, a locked nucleic acid (LNA), auniversal base, or combinations thereof, and can include stem-loop andstem-less reporter probe configurations. Certain reporter probes aresingly-labeled, while other reporter probes are doubly-labeled. Dualprobe systems that comprise FRET between adjacently hybridized probesare within the intended scope of the term reporter probe. In certainembodiments, a reporter probe comprises a fluorescent reporter group anda quencher (including without limitation dark quenchers and fluorescentquenchers). Some non-limiting examples of reporter probes includeTaqMan® probes; Scorpion probes (also referred to as scorpion primers);Lux™ primers; FRET primers; Eclipse® probes; molecular beacons,including but not limited to FRET-based molecular beacons, multicolormolecular beacons, aptamer beacons, PNA beacons, and antibody beacons;labeled PNA clamps, labeled PNA openers, labeled LNA probes, and probescomprising nanocrystals, metallic nanoparticles and similar hybridprobes (see, e.g., Dubertret et al., Nature Biotech. 19:365-70, 2001;Zelphati et al., BioTechniques 28:304-15, 2000). In certain embodiments,reporter probes further comprise minor groove binders including but notlimited to TaqMan®MGB probes and TaqMan®MGB-NFQ probes (both fromApplied Biosystems). In certain embodiments, reporter probe detectioncomprises fluorescence polarization detection (see, e.g., Simeonov andNikiforov, Nucl. Acids Res. 30:e91, 2002).

The term “reporter group” is used in a broad sense herein and refers toany identifiable tag, label, or moiety. The ordinarily skilled artisanwill appreciate that many different species of reporter groups can beused in the present teachings, either individually or in combinationwith one or more different reporter group. In certain embodiments, areporter group emits a fluorescent, a chemiluminescent, abioluminescent, a phosphorescent, or an electrochemiluminescent signal.Some non-limiting examples of reporter groups include fluorophores,radioisotopes, chromogens, enzymes, antigens including but not limitedto epitope tags, semiconductor nanocrystals such as quantum dots, heavymetals, dyes, phosphorescence groups, chemiluminescent groups,electrochemical detection moieties, binding proteins, phosphors, rareearth chelates, transition metal chelates, near-infrared dyes,electrochemiluminescence labels, and mass spectrometer-compatiblereporter groups, such as mass tags, charge tags, and isotopes (see,e.g., Haff and Smirnov, Nucl. Acids Res. 25:3749-50, 1997; Xu et al.,Anal. Chem. 69:3595-3602, 1997; Sauer et al., Nucl. Acids Res. 31:e63,2003).

The term reporter group also encompasses an element of multi-elementreporter systems, including without limitation, affinity tags such asbiotin:avidin, antibody:antigen, and the like, in which one elementinteracts with one or more other elements of the system in order toeffect the potential for a detectable signal. Some non-limiting examplesof multi-element reporter systems include an oligonucleotide comprisinga biotin reporter group and a streptavidin-conjugated fluorophore, orvice versa; an oligonucleotide comprising a DNP reporter group and afluorophore-labeled anti-DNP antibody; and the like. Detailed protocolsfor attaching reporter groups to nucleic acids can be found in, amongother places, Hermanson, Bioconjugate Techniques, Academic Press, SanDiego, 1996; Current Protocols in Nucleic Acid Chemistry, Beaucage etal., eds., John Wiley & Sons, New York, N.Y. (2000), includingsupplements through April 2005; and Haugland, Handbook of FluorescentProbes and Research Products, 9^(th) ed., Molecular Probes, 2002.

Multi-element interacting reporter groups are also within the intendedscope of the term reporter group, such as fluorophore-quencher pairs,including without limitation fluorescent quenchers and dark quenchers(also known as non-fluorescent quenchers). A fluorescent quencher canabsorb the fluorescent signal emitted from a fluorophore and afterabsorbing enough fluorescent energy, the fluorescent quencher can emitfluorescence at a characteristic wavelength, e.g., fluorescent resonanceenergy transfer (FRET). For example without limitation, the FAM™-TAMRA™dye pair can be illuminated at 492 nm, the excitation peak for FAM™ dye,and emit fluorescence at 580 nm, the emission peak for TAMRA™ dye. Adark quencher, appropriately paired with a fluorescent reporter group,absorbs the fluorescent energy from the fluorophore, but does not itselffluoresce. Rather, the dark quencher dissipates the absorbed energy,typically as heat. Some non-limiting examples of dark or nonfluorescentquenchers include Dabcyl, Black Hole Quenchers, Iowa Black, QSY-7,AbsoluteQuencher, Eclipse® non-fluorescent quencher, metal clusters suchas gold nanoparticles, and the like. Certain dual-labeled probescomprising fluorophore-quencher pairs can emit fluorescence when themembers of the pair are physically separated, for example but withoutlimitation, nuclease probes such as TaqMan® probes. Other dual-labeledprobes comprising fluorophore-quencher pairs can emit fluorescence whenthe members of the pair are spatially separated, for example but notlimited to hybridization probes such as molecular beacons or extensionprobes such as Scorpion™ primers. Fluorophore-quencher pairs are wellknown in the art and used extensively for a variety of reporter probes(see, e.g., Yeung et al., BioTechniques 36:266-75, 2004; Dubertret etal., Nat. Biotech. 19:365-70, 2001; and Tyagi et al., Nat. Biotech.18:1191-96, 2000).

The term “nucleic acid dye” as used herein refers to a fluorescentmolecule that is specific for a double-stranded polynucleotide or thatemits a substantially greater fluorescent signal when associated with adouble-stranded polynucleotide than with a single-strandedpolynucleotide. Typically nucleic acid dye molecules associate withdouble-stranded segments of polynucleotides by intercalating between thebase pairs of the double-stranded segment, by binding in the major orminor grooves of the double-stranded segment, or both. Non-limitingexamples of nucleic acid dyes include ethidium bromide, DAPI, Hoechstderivatives including without limitation Hoechst 33258 and Hoechst33342, intercalators comprising a lanthanide chelate (for example butnot limited to a nalthalene diimide derivative carrying two fluorescenttetradentate β-diketone-Eu3+ chelates (NDI-(BHHCT-Eu³⁺)₂), see, e.g.,Nojima et al., Nucl. Acids Res. Supplement No. 1, 105-06 (2001)),ethidium bromide, and certain unsymmetrical cyanine dyes such as SYBR®Green, SYBR® Gold, PicoGreen®, and BOXTO.

In certain embodiments, detecting comprises an instrument, i.e., usingan automated or semi-automated detecting device that can, but need notcomprise a computer algorithm. In certain embodiments, a detectinginstrument comprises or is coupled to a device for graphicallydisplaying the intensity of an observed or measured parameter of anextension product or its surrogate on a graph, monitor, electronicscreen, magnetic media, scanner print-out, or other two- orthree-dimensional display and/or recording the observed or measuredparameter. In certain embodiments, the detecting step is combined withor is a continuation of at least one separating step, for example butnot limited to a capillary electrophoresis instrument comprising atleast one fluorescent scanner and at least one graphing, recording, orreadout component; a chromatography column coupled with an absorbancemonitor or fluorescence scanner and a graph recorder; a chromatographycolumn coupled with a mass spectrometer comprising a recording and/or adetection component; or a microarray with a data recording device suchas a scanner or CCD camera. In certain embodiments, the detecting stepis combined with an amplifying step, for example but not limited to,real-time analysis such as Q-PCR. Exemplary systems for performing adetecting step include the ABI PRISM® Genetic Analyzer instrumentseries, the ABI PRISM® DNA Analyzer instrument series, the ABI PRISM®Sequence Detection Systems instrument series, and the Applied BiosystemsReal-Time PCR instrument series (all from Applied Biosystems); andmicroarrays and related software such as the Applied Biosystemsmicroarray and Applied Biosystems 1700 Chemiluminescent MicroarrayAnalyzer and other commercially available microarray and analysissystems available from Affymetrix, Agilent, among others (see also Gerryet al., J. Mol. Biol. 292:251-62, 1999; De Bellis et al., Minerva Biotec14:247-52, 2002; and Stears et al., Nat. Med. 9:140-45, includingsupplements, 2003) or bead array platforms (Illumina, San Diego,Calif.). Exemplary software includes GeneMapper™ Software, GeneScan®Analysis Software, and Genotyper® software (all from AppliedBiosystems).

In certain embodiments, an RNA molecule can be detected and quantifiedbased on the mass-to-charge ratio (m/z) of at least a part of anamplified product and/or a ligated product. For example, in someembodiments, a primer or an adapter comprises a massspectrometry-compatible reporter group, including without limitation,mass tags, charge tags, cleavable portions, or isotopes that areincorporated into an amplified product and can be used for massspectrometer detection (see, e.g., Haff and Smirnov, Nucl. Acids Res.25:3749-50, 1997; and Sauer et al., Nucl. Acids Res. 31:e63, 2003). Anamplified product can be detected by mass spectrometry allowing thepresence or absence of the corresponding RNA molecule to be determined.In some embodiments, a primer or an adaptor comprises a restrictionenzyme site, a cleavable portion, or the like, to facilitate release ofa part of an amplified product for detection. In certain embodiments, amultiplicity of amplified products are separated by liquidchromatography or capillary electrophoresis, subjected to ESI or toMALDI, and detected by mass spectrometry. Descriptions of massspectrometry can be found in, among other places, The Expanding Role ofMass Spectrometry in Biotechnology, Gary Siuzdak, MCC Press, 2003.

In certain embodiments, surrogates such as a reporter probe or a cleavedportion of a reporter probe are detected, directly or indirectly. Forexample but not limited to, hybridizing an amplified product to areporter probe comprising a quencher, including without limitation, amolecular beacon, including stem-loop and stem-free beacons, a TaqMan®probe or other nuclease probe, a LightSpeed™ PNA probe, or a microarraycapture probe. In certain embodiments, the hybridization occurs when themolecular beacon and the amplified product are free in solution and adetectable signal or a detectably different signal is emitted. In otherembodiments, an amplified product hybridizes to or is bound to a solidsurface such as a microarray and a detectable signal or a detectablydifferent signal is emitted (see, e.g., EviArrays™ and EviProbes™,Evident Technologies).

In certain embodiments, detecting comprises measuring or quantifying thedetectable signal of a reporter group or the change in a detectablesignal of a reporter group, typically due to the presence of anamplified product. For illustration purposes but not as a limitation, anunhybridized reporter probe may emit a low level, but detectable signalthat quantitatively increases when hybridized with the amplifiedproduct, including without limitation, certain molecular beacons, LNAprobes, PNA probes, and light-up probes (see, e.g., Svanik et al.,Analyt. Biochem. 281:26-35, 2000; Nikiforov and Jeong, Analyt. Biochem.275:248-53, 1999; and Simeonov and Nikiforov, Nucl. Acids Res. 30:e91,2002). In certain embodiments, detecting comprises measuringfluorescence polarization.

In some embodiments, determining whether a particular RNA molecule ispresent in a sample comprises evaluating an internal standard or acontrol sequence, such as a standard curve for the corresponding targetregion, an internal size standard, or combinations thereof. In someembodiments, a control sequence or an internal reference dye is employedto account for lane-to-lane, capillary-to-capillary, and/orassay-to-assay variability. In certain embodiments, an internal controlsequence comprises an unrelated nucleic acid that is amplified inparallel to validate the amplification reaction or the detectiontechnique.

Those in the art understand that the detection techniques employed aregenerally not limiting. Rather, a wide variety of detection methods arewithin the scope of the disclosed methods and kits, provided that theyallow the presence or absence of an RNA molecule in the sample to bedetermined.

In some embodiments, the disclosed methods and kits comprise amicrofluidics device, “lab on a chip”, or micrototal analytical system(μTAS). In some embodiments, sample preparation is performed using amicrofluidics device. In some embodiments, an amplification reaction isperformed using a microfluidics device. In some embodiments, asequencing or Q-PCR reaction is performed using a microfluidic device.In some embodiments, the nucleotide sequence of at least a part of anamplified product is obtained using a microfluidics device. In someembodiments, detecting comprises a microfluidic device, includingwithout limitation, a TaqMan® Low Density Array (Applied Biosystems).Descriptions of exemplary microfluidic devices can be found in, amongother places, Published PCT Application Nos. WO/0185341 and WO04/011666; Kartalov and Quake, Nucl. Acids Res. 32:2873-79, 2004; andFiorini and Chiu, BioTechniques 38:429-46, 2005.

Sequencing:

In some embodiments, the sequence of at least part of the amplifiedproduct is determined thereby detecting the RNA molecule of interest.The term “sequencing” is used in a broad sense herein and refers to anytechnique known in the art that allows the order of at least someconsecutive nucleotides in at least part of a RNA to be identified,including without limitation at least part of an extension product or avector insert. Some non-limiting examples of sequencing techniquesinclude Sanger's dideoxy terminator method and the chemical cleavagemethod of Maxam and Gilbert, including variations of those methods;sequencing by hybridization, for example but not limited to,hybridization of amplified products to a microarray or a bead, such as abead array; pyrosequencing (see, e.g., Ronaghi et al., Science281:363-65, 1998); and restriction mapping. Some sequencing methodscomprise electrophoreses, including without limitation capillaryelectrophoresis and gel electrophoresis; mass spectrometry; and singlemolecule detection. In some embodiments, sequencing comprises directsequencing, duplex sequencing, cycle sequencing, single-base extensionsequencing (SBE), solid-phase sequencing, or combinations thereof. Insome embodiments, sequencing comprises an detecting the sequencingproduct using an instrument, for example but not limited to an ABIPRISM® 377 DNA Sequencer, an ABI PRISM® 310, 3100, 3100-Avant, 3730, or3730×1 Genetic Analyzer, an ABI PRISM® 3700 DNA Analyzer, or an AppliedBiosystems SOLiD™ System (all from Applied Biosystems), a GenomeSequencer 20 System (Roche Applied Science), or a mass spectrometer. Incertain embodiments, sequencing comprises emulsion PCR (see, e.g.,Williams et al., Nature Methods 3(7):545-50, 2006.) In certainembodiments, sequencing comprises a high throughput sequencingtechnique, for example but not limited to, massively parallel signaturesequencing (MPSS). Descriptions of MPSS can be found, among otherplaces, in Zhou et al., Methods of Molecular Biology 331:285-311, HumanaPress Inc.; Reinartz et al., Briefings in Functional Genomics andProteomics, 1:95-104, 2002; Jongeneel et al., Genome Research15:1007-14, 2005. In some embodiments, sequencing comprisesincorporating a dNTP, including without limitation a dATP, a dCTP, adGTP, a dTTP, a dUTP, a dITP, or combinations thereof and includingdideoxyribonucleotide versions of dNTPs, into an amplified product.

Further exemplary techniques that are useful for determining thesequence of at least a portion of a nucleic acid molecule include,without limitation, emulsion-based PCR followed by any suitablemassively parallel sequencing or other high-throughput technique. Insome embodiments, determining the sequence of at least a part of anamplified product to detect the corresponding RNA molecule comprisesquantitating the amplified product. In some embodiments, sequencing iscarried out using the SOLiD™ System (Applied Biosystems) as describedin, for example, PCT patent application publications WO 06/084132entitled “Reagents, Methods, and Libraries For Bead-Based Sequencing andWO07/121,489 entitled “Reagents, Methods, and Libraries for Gel-FreeBead-Based Sequencing.” In some embodiments, quantitating the amplifiedproduct comprises real-time or end-point quantitative PCR or both. Insome embodiments, quantitating the amplified product comprisesgenerating an expression profile of the RNA molecule to be detected,such as an mRNA expression profile or a miRNA expression profile. Incertain embodiments, quantitating the amplified product comprises one ormore 5′-nuclease assays, for example but not limited to, TaqMan® GeneExpression Assays and TaqMan® miRNA Assays, which may comprise amicrofluidics device including without limitation, a low density array.Any suitable expression profiling technique known in the art may beemployed in various embodiments of the disclosed methods.

Those in the art will appreciate that the sequencing method employed isnot typically a limitation of the present methods. Rather, anysequencing technique that provides the order of at least someconsecutive nucleotides of at least part of the corresponding amplifiedproduct or RNA to be detected or at least part of a vector insertderived from an amplified product can typically be used in the currentmethods. Descriptions of sequencing techniques can be found in, amongother places, McPherson, particularly in Chapter 5; Sambrook andRussell; Ausubel et al.; Siuzdak, The Expanding Role of MassSpectrometry in Biotechnology, MCC Press, 2003, particularly in Chapter7; and Rapley. In some embodiments, unincorporated primers and/or dNTPsare removed prior to a sequencing step by enzymatic degradation,including without limitation exonuclease I and shrimp alkalinephosphatase digestion, for example but not limited to the ExoSAP-IT®reagent (USB Corporation). In some embodiments, unincorporated primers,dNTPs, and/or ddNTPs are removed by gel or column purification,sedimentation, filtration, beads, magnetic separation, orhybridization-based pull out, as appropriate (see, e.g., ABI PRISM®Duplex™ 384 Well F/R Sequence Capture Kit, Applied Biosystems P/N4308082).

Those in the art will appreciate that, in certain embodiments, the readlength of the sequencing/resequencing technique employed may be a factorin the size of the RNA molecules that can effectively be detected (see,e.g., Kling, Nat. Biotech. 21(12):1425-27). In some embodiments, theamplified products generated from the RNA molecules from a first sampleare labeled with a first identification sequence (sometimes referred toas a “barcode” herein) or other marker, the amplified products generatedfrom the RNA molecules from a second sample are labeled with a secondidentification sequence or second marker, and the amplified productscomprising the first identification sequence and the amplified productscomprising the second identification sequence are pooled prior todetermining the sequence of the corresponding RNA molecules in thecorresponding samples. In certain embodiments, three or more differentRNA libraries, each comprising a identifier sequence that is specific tothat library, are combined. In some embodiments, a first adaptor, asecond adaptor, a forward primer, a reverse primer, or combinationsthereof, comprise an identification sequence or the complement of anidentification sequence. In certain embodiments, the identificationsequence comprises one of (i) 5′-AAGCCC, (ii) 5′-CACACC, (iii)5′-CCCCTT, (iv) 5′-CATCGG, (v) 5-TCGTTG, (vi) 5′-GGGCAC, (vii)5′-CCAGAC, (viii) 5′-CTCCGT, (ix) 5′-CCCTTC, (x) 5′-GCGGTC, or thecomplement of any one of these sequences (i)-(x). In some embodiments, areverse primer comprises a sequence of SEQ ID NO:6 or SEQ ID NO:15 toSEQ ID NO:23 as described in Example 11.

Libraries:

The present teachings provide compositions, methods and kits fordetecting an RNA molecule. According to certain embodiments, a librarycomprising a multiplicity of different amplified product species isgenerated wherein at least one species of amplified product correspondsto one species of small RNA present in the sample.

According to certain illustrative embodiments of the instant teachings,for example, a sample comprising a multiplicity of small RNA species, amultiplicity of mRNA species, or both, is combined with a multiplicityof different first adaptor species, a multiplicity of different secondadaptor species, and a polypeptide comprising double-strand specific RNAligase activity to form a ligation reaction composition. In someembodiments, the mRNA is fragmented, depleted of undesired nucleic acidspecies (for example but not limited to, rRNA, high copy number mRNAs orgenomic DNA), or depleted and fragmented. The ligation reactioncomposition is incubated under conditions suitable for at least some ofthe adaptor species to hybridize with corresponding RNA molecules. It isto be understood that the process of (i) combining the adaptor specieswith the sample containing RNA, (ii) incubating to allow the adaptors toanneal with a corresponding RNA molecule, then (iii) adding the ligaseto the reaction composition is within the intended scope of forming theligation reaction composition and incubating, unless expressly statedotherwise.

The multiplicity of different adaptor species typically comprise sets ofRNA/DNA oligonucleotides with single-stranded degenerate sequence at oneend and in certain embodiments, a defined sequence at or near the otherend that may serve as a binding site for amplification primers orreporter probes, sample identification (for example but not limited topooling libraries generated from different starting materials andsubsequently identifying the source of the amplified library), and/orsequencing of subsequently generated amplified products. In certainembodiments, hybridizing sample with Adaptor Mix A will yield amplifiedproducts suitable for SOLiD™ sequencing from the 5′ ends of the sequencecorresponding to the RNA molecule within the amplified product.Conversely, hybridization with Adaptor Mix B yields amplified productssuitable for SOLiD™ sequencing from the 3′ ends. A polypeptidecomprising double-strand specific RNA ligase activity is then added tothe mixture to ligate the hybridized adaptors to the small RNAmolecules.

The ligation reaction composition is combined with a DNA polymerasecomprising RNA-dependent DNA polymerase activity and the ligated productis reverse transcribed to generate cDNA. This reverse transcribedproduct is combined with RNase H to digest at least some of the smallRNA or fragmented mRNA from the RNA/cDNA duplexes, generatingamplification templates. Those in the art will appreciate that theconcentration of unligated adaptors and adaptor by-products is alsodecreased during the ribonuclease digestion process. At this point,reactions contain cDNA copies of the RNA molecules in the sample. Tomeet the amplified product input requirements for certain sequencingtechniques, and in some embodiments to append identifier sequences tothe amplified products, the reverse transcribed products may beamplified using appropriate primer sets, wherein at least one forwardprimer, at least one reverse primer, or both may comprise one or moreidentifier sequences, and a number of PCR amplification cycles whereindetection is in a linear range when plotted vs. cycle number (˜12-15 or˜12-18 cycles of PCR). Those in the art will appreciate that limitingthe cycle number minimizes the synthesis of spurious PCR products andpreserves the integrity of the RNA profile of the sample. In certainembodiments, at least one forward primer, at least one reverse primer,or at least one forward and at least one reverse primer comprise one ormore identifier sequences. Certain embodiments comprise use of ten setsof PCR primers that have the same nucleotide sequence, except for a 6 bp“barcode” identifier sequence on the 3′ (reverse) primer that isspecific to that reverse primer species.

In certain embodiments, the amplification reaction products aresubjected to size selection, for example but not limited to, gelelectrophoresis, to concentrate the amplified products in a desired sizerange and remove PCR by-products. Appropriately size selected amplifiedproducts can be used in the SOLiD™ Sequencing System (AppliedBiosystems) workflow at the emulsion PCR (ePCR) step where the amplifiedproducts are attached to beads, further amplified using ePCR andultimately sequenced, which allows the presence, absence, and/orquantity of various RNA molecules in the sample to be determined.

Those in the art will appreciate that, in certain circumstances, anamplified product and/or a ligated product can serve as a surrogate forthe corresponding RNA molecule and that by detecting the amplifiedproduct, the ligated product, or both, the RNA molecule is indirectlydetected and that such detection is within the scope of the currentteachings.

Kits:

Kits for performing certain of the instant methods are also disclosed.Certain kit embodiments include first adaptors, second adaptors, apolypeptide comprising double-strand specific RNA ligase activity,reverse transcriptase, ribonuclease H(RNase H), DNA polymerase, primers,or combinations thereof. In some embodiments, kits further comprise anagent for removing 5′ phosphates from RNA, for example but not limitedto, tobacco acid pyrophosphatase.

The instant teachings also provide kits designed to expedite performingcertain of the disclosed methods. Kits may serve to expedite theperformance of certain disclosed methods by assembling two or morecomponents required for carrying out the methods. In certainembodiments, kits contain components in pre-measured unit amounts tominimize the need for measurements by end-users. In some embodiments,kits include instructions for performing one or more of the disclosedmethods. In some embodiments, the kit components are optimized tooperate in conjunction with one another.

In certain embodiments, kits comprise at least one first adaptorspecies, at least one second adaptor species, a polypeptide comprisingdouble-strand specific RNA ligase activity, a DNA polymerase, includingwithout limitation, a RNA-directed DNA polymerase, a DNA-directed DNApolymerase, or a DNA polymerase comprising both RNA-directed andDNA-directed DNA polymerase activities, ribonuclease H, or combinationsthereof. In certain embodiments, the ligase comprises bacteriophage T4RNA ligase 2 (Rnl2) or a ligase from the Rnl2 family. In someembodiments, the first adaptor, the second adaptor, or both the firstadaptor and the second adaptor comprise a single-stranded portioncomprising degenerate sequences.

In certain embodiments, a kit comprises a plurality of first adaptorspecies, wherein each first adaptor species comprises a differentdegenerate sequence, a plurality of second adaptor species, wherein eachsecond adaptor species comprises a different degenerate sequence, aligase of the Rnl2 family, a RNA-directed DNA polymerase, a plurality ofdifferent first primer species, a DNA-directed DNA polymerase and RNaseH (EC 3.1.26.4). In some embodiments, the kit further comprises tobaccoacid pyrophosphatase.

In certain embodiments, a kit comprises a plurality of first adaptorspecies, wherein at least some of the first adaptor species comprise adegenerate sequence, a plurality of second adaptor species, wherein atleast some of the second adaptor species comprise a degenerate sequence,a polypeptide comprising double-strand specific RNA ligase activity, aDNA polymerase, at least one primer species, and a ribonuclease. In someembodiments, the DNA polymerase of the kit comprises an RNA-dependentDNA polymerase and a DNA-dependent DNA polymerase. In addition, the kitmay comprise tobacco acid pyrophosphatase.

In certain embodiments, kits further comprise a forward amplificationprimer and a reverse amplification primer. In some embodiments, aforward primer, a reverse primer, or both a forward and a reverse primercomprise a universal priming sequence or the complement of a universalpriming sequence. In some embodiments, kits comprise a forward primer, areverse primer, or a forward primer and a reverse primer that furthercomprises a reporter group. In some such embodiments, the reporter groupof a forward primer of a primer pair is different from the reportergroup of the reverse primer of the primer pair. In some embodiments,kits further comprise at least one of: a reporter probe, a nucleic aciddye, a reporter group, or combinations thereof. In some embodiments,kits further comprise a control sequence, for example but not limited toan internal standard sequence such as a housekeeping gene or apolynucleotide ladder comprising molecular size or weight standards.

In certain kit embodiments a first adaptor, a second adaptor, a forwardprimer, a reverse primer, or combinations thereof, comprise anidentification sequence or the complement of an identification sequence.In certain embodiments, the identification sequence comprises one of (i)5′-AAGCCC, (ii) 5′-CACACC, (iii) 5′-CCCCTT, (iv) 5′-CATCGG, (v)5-TCGTTG, (vi) 5′-GGGCAC, (vii) 5′-CCAGAC, (viii) 5′-CTCCGT, (ix)5′-CCCTTC, (x) 5′-GCGGTC, or the complement of any one of thesesequences (i)-(x). In some embodiments, a reverse primer comprises thesequence of one of SEQ ID NO:6 and SEQ ID NO:15 to SEQ ID NO:23. In somekit embodiments, mixtures of forward primers and reverse primers areprovided. In some embodiments, kits provide a plurality of primermixtures, for example but not limited to, at least two of: (i) a primermixture comprising a first forward primer and a first reverse primer,wherein the first reverse primer comprises a first identificationsequence; (ii) a primer mixture comprising the first forward primer anda second reverse primer, wherein the second reverse primer comprises asecond identification sequence; (iii) a primer mixture comprising thefirst forward primer and a third reverse primer, wherein the thirdreverse primer comprises a third identification sequence; (iv) a primermixture comprising the first forward primer and a fourth reverse primer,wherein the fourth reverse primer comprises a fourth identificationsequence; (v) a primer mixture comprising the first forward primer and afifth reverse primer, wherein the fifth reverse primer comprises a fifthidentification sequence; (vi) a primer mixture comprising the firstforward primer and a sixth reverse primer, wherein the sixth reverseprimer comprises a sixth identification sequence; (vii) a primer mixturecomprising first forward primer and a seventh reverse primer, whereinthe seventh reverse primer comprises a seventh identification sequence;(viii) a primer mixture comprising first forward primer and an eighthreverse primer, wherein the eighth reverse primer comprises an eighthidentification sequence; (ix) a primer mixture comprising the firstforward primer and a ninth reverse primer, wherein the ninth reverseprimer comprises a ninth identification sequence; and (x) a primermixture comprising the first forward primer and a tenth reverse primer,wherein the tenth reverse primer comprises a tenth identificationsequence.

Some kit embodiments comprise at least one adaptor mix, wherein eachadaptor mix comprises a first adaptor and a second adaptor; at least onepolypeptide comprising double-strand specific RNA ligase activity; areverse transcriptase (RNA-directed DNA polymerase); a DNA polymerase(DNA-directed DNA polymerase); a ribonuclease; a mixture ofdeoxyribonucleotide triphosphates (dNTPs); and at least oneamplification primer mix, wherein each amplification primer mixcomprises a forward primer and reverse primer. In some embodiments, theRNA-directed DNA polymerase and the DNA-directed DNA polymerase compriseeither an RNA-directed DNA polymerase that possesses DNA-directed DNApolymerase activity under certain reaction conditions or a DNA-directedDNA polymerase that possesses RNA-directed DNA polymerase activity undercertain reaction conditions, for example but not limited to, Tth DNApolymerase and DNA polymerase I from Carboxydothermus hydrogenoformans.In some kit embodiments, the DNA-dependent DNA polymerase comprises TaqDNA polymerase, including enzymatically active mutants and variantsthereof, for example but not limited to, AmpliTaq® DNA polymerase andAmpliTaq Gold® DNA polymerase (Applied Biosystems). In some embodiments,the ribonuclease comprises ribonuclease H(RNase H). Some kit embodimentscomprise at least one control RNA molecule, for example but not limitedto, at least one positive control RNA molecule, at least one negativecontrol RNA molecule, or both.

Solid Supports:

In certain embodiments, the disclosed methods and kits comprise a solidsupport. In some embodiments, a solid support is used in a separatingand/or detecting step, for example but not limited to, for purifyingand/or analyzing amplification products. Non-limiting examples of solidsupports include, agarose, sepharose, polystyrene, polyacrylamide,glass, membranes, silica, semiconductor materials, silicon, organicpolymers; optically identifiable micro-cylinders; biosensors comprisingtransducers; appropriately treated or coated reaction vessels andsurfaces, for example but not limited to, micro centrifuge or reactiontubes, wells of a multiwell microplate, and glass, quartz or plasticslides and/or cover slips; and beads, for example but not limited tomagnetic beads, paramagnetic beads, polymer beads, metallic beads,dye-impregnated or labeled beads, coated beads, glass beads,microspheres and nanospheres. Those in the art will appreciate that anynumber of solid supports may be employed in the disclosed methods andkits and that the shape and composition of the solid support isgenerally not limiting.

The current teachings, having been described above, may be betterunderstood by reference to examples. The following examples are intendedfor illustration purposes only, and should not be construed as limitingthe scope of the teachings herein in any way.

Example 1 Generation of Amplified Products Using Double Strand-SpecificRNA Ligase Rnl2

Each of three RNA samples:

-   -   total RNA from human placenta (100 μg, Ambion P/N AM7950),    -   synthetic miRNA molecules (mirVana™ miRNA Reference Panel v.9.1,        100 fmol, Ambion P/N 4388891, an equimolar pool of synthetic RNA        oligonucleotides representing most of the human, mouse, and rat        miRNA sequences in miRBase Sequence Database Version 9.1), and    -   flashPAGE™ Fractionation System-purified total RNA (human        placenta) from 5 μg total RNA,        was mixed with 3 μL hybridization solution (300 mM NaCl, 20 mM        Tris-HCl pH 8.0, 2 mM EDTA) and 2 μL Adaptor oligo mix        containing oligonucleotides as follows:

3.1 μM (5′-upper, i.e., first oligonucleotide) SEQ ID NO: 15′-CCUCUCUAUGGGCAGUCGGUGAU-3′,;6.1 μM (5′-lower, i.e., second oligonucleotide) SEQ ID NO: 25′-NNNNNNATCACCGACTGCCCATAGAGAGG-3′,;3.1 μM (3′-upper, i.e., third oligonucleotide) SEQ ID NO: 35′-PO₄-CGCCTTGGCCGTACAGCAG-3′,; and6.1 μM (3′-lower, i.e., fourth oligonucleotide) SEQ ID NO: 4)5′-CTGCTGTACGGCCAAGGCGNNNNNN-3′;

where “N” represents degenerate bases (A, C, G or T) mixed in a25:25:25:25 ratio.

The lyophilized oligonucleotides were resuspended to a stockconcentration of 100 μM in nuclease-free water (Ambion P/N AM9937) anddiluted accordingly. The RNA mixture was heated at 65° C. for 10 minutesfollowed by 5 minutes at 16° C. to hybridize the adaptors to the smallRNA in the sample.

The hybridized RNA/Adaptors were then mixed with 10 μL Ligation Buffer(100 mM Tris-HCl pH 7.5, 20 mM MgCl₂, 20 mM dithiothreitol, 2 mM ATP,and 40% (w/v) PEG-8000) and ligated with 20 units of T4 RNA ligase 2(NEB, Ipswich, Mass.) in a total of 20 μL for 16 hours at 16° C. Inaddition, a minus ligase control was prepared for the total RNA sampleusing nuclease-free water in place of the ligase enzyme.

The ligated products were reverse transcribed by incubating for 30minutes at 42° C. with 200 units of ArrayScript™ reverse transcriptase(Ambion P/N AM2048), 4 μL 10×RT buffer (provided with the ArrayScript™enzyme) and 2 μL 2.5 mM dNTP mix (Ambion P/N AM8228G) in a total volumeof 40 μL. A minus RT control was included for the total RNA sample usingnuclease-free water in place of the reverse transcriptase.

Excess RNA byproducts were removed by incubating a 10 μL aliquot of thesample at 37° C. for 30 minutes with 10 units of RNase H (Ambion P/NAM2292) and the RNase-treated cDNA was amplified by PCR. The 50 μL PCRreactions contained the following:

38.5 μL nuclease-free water (Ambion P/N AM9937),    5 μL GeneAmp ®10X PCR Buffer I (Applied Biosystems P/N N8080246),   4 μL 2.5 mM dNTP mix (Ambion P/N AM8228G), 0.5 μL 50 μM forward PCR primer: (SEQ ID NO: 5)(5′-CCACTACGCCTCCGCTTTCCTCTCTATGGGCAGTCGGTGAT-3′, 0.5 μL 50 μM reverse PCR primer: (SEQ ID NO: 6)(5′-CTGCCCCGGGTTCCTCATTCTCTCCAGACCTGCTGTACGGCCAA GGCG-3′),   1 μL AmpliTaq ® DNA Polymerase (Applied Biosystems P/N N8080246), and 0.5 μL cDNA sample.

The PCR conditions were as follows: initial denaturation at 95° C. for 5minutes, followed by 15 cycles of 95° C. for 30 seconds (denaturation),62° C. for 30 seconds (annealing), and 72° C. for 30 seconds(extension). A final extension at 72° C. for 7 minutes followed.

Ten μL of each sample was mixed 1:1 with Gel Loading Buffer II (AmbionP/N AM8547) and the entire volume was loaded onto a 1.0 mm 6%polyacrylamide gel and electrophoresed for approximately 45 minutes at180 volts constant in 1× tris-borate EDTA running buffer (Ambion P/NAM9863). The gel was removed from the cassette and stained in 1×SYBR®Gold nucleic acid gel stain (Invitrogen P/N S11494) in 1×TBE for 5minutes. The gel was imaged using the Alpha Innotech Fluor Chem SPimager and the image processed with AlphaEase® FC software version6.0.0.

As seen in FIG. 5, the amplified ligation products (amplified product)are shown by bracket A; arrow B denotes undesired by-products of thereaction that were amplified in the amplification reaction; bracket Cindicates the residual unligated adaptors and primers in theamplification reaction composition.

Note that the forward PCR primer sequence contains SEQ ID NO:1, thesequence of the first oligonucleotide (with T substituted for U),beginning at nucleotide 19 of the forward primer. Note also that thereverse primer sequence contains SEQ ID NO:4, the sequence of the fourtholigonucleotide (with T substituted for U), beginning at nucleotide 30of the reverse primer. Therefore, with this construction, a detectedsmall RNA will have a length equal to a gel fragment size as seen inFIG. 5, for example, less the total of the lengths of the primers.

Example 2 Generation of Amplified Product Using Various Double-StrandSpecific Ligases

Total RNA from human placenta (500 ng, Ambion P/N AM7950) was mixed with3 μL Hybridization solution and 2 μL Adaptor oligo mix and hybridized asin Example 1. The hybridized RNA/Adaptors were then mixed with 10 μLLigation Buffer (100 mM Tris-HCl pH 7.5, mM MgCl₂, 20 mM dithiothreitol,2 mM ATP, and 40% (w/v) PEG-8000) and ligated with 10 units of eitherbacteriophage T4 RNA ligase 2, bacteriophage T4 RNA ligase 1 (Ambion P/NAM2140), bacteriophage T4 DNA ligase (Ambion P/N AM2134), or a 1:1mixture of T4 RNA ligase I and T4 DNA ligase in a total of 20 μL for 16hours at 16° C. In addition, a minus ligase control was prepared usingnuclease-free water in place of the ligase enzyme.

The ligated products were reverse transcribed, treated with RNase H andamplified as in Example 1. Ten μL of each sample was mixed 1:1 with GelLoading Buffer II (Ambion P/N AM8547) and the entire volume was loadedonto a 1.0 mm 6% polyacrylamide gel (Invitrogen P/N EC6265BOX) andelectrophoresed for approximately 45 minutes at 180 volts constant in 1×tris-borate EDTA running buffer (Ambion P/N AM9863). The gel was removedfrom the cassette and stained in 1×SYBR® Gold nucleic acid gel stain(Invitrogen P/N S11494) in 1×TBE for 5 minutes. The gel was imaged usingthe Alpha Innotech Fluor Chem SP imager and the image processed withAlphaEase® FC software ver. 6.0.0. Bands were compared to a 10 bp DNAladder (100 ng; Invitrogen P/N 10821-015).

As seen in FIG. 6, the amplified ligation products (amplified product)are shown by bracket A; arrow B denotes undesired by-products of thereaction that were amplified; and bracket C indicates the residualunligated adaptors and primers in the amplification reactioncomposition. Surprisingly, when the amplified products generated usingligase Rnl2 (lane 2), ligase Rnl1 (lane 4), ligase Dnl (lane 6), or acombination of both Rnl1 and Dnl (lane 8) are compared, a differentamplified product profile is observed. Amplified product in the 110 basepair (bp) to 130 bp size range (arrow A), which in this illustrativeembodiment is in the size range expected for amplified miRNA, isobserved when Rnl2 ligase is used, but not when Rnl1 or Dnl are used,either alone or in combination. Additionally, a prominent band ofamplified product of approximately 100 bp (migrating slightly above B inFIG. 6) appears in all lanes corresponding to Rnl1, Dnl or both (withand without reverse transcriptase), but this band is not observed whenRnl2 is used. This difference in amplified products generated with Rnl2in comparison to the amplified products generated using two differentligases either alone or in combination in parallel reactions issurprising and unexpected.

Example 3 Generation of Barcoded Amplified Products for Sequencing

Small RNA is obtained from HeLa cells using the mirVana™ miRNA IsolationKit (AM1560, Ambion) according to the manufacturer's Instruction Manualfollowing the procedures for total RNA isolation.

Ligation reaction compositions were prepared as follows. Hybridizationmixtures were prepared in 0.2 mL RNase-free thin-walled PCR tubescomprising for each reaction: 2 μL adaptor mix (first adaptor and secondadaptor), 3 μL 2× hybridization solution (300 mM NaCl, 20 mM Tris, 2 mMEDTA, final pH 8.0), 1-3 μL RNA molecule solution from mirVana™ miRNAIsolation Kit (containing 1000 nanograms (ng) RNA) or 1000 ng positivecontrol (FirstChoice® Total RNA: Human Placenta; AM7950, Ambion), and0-2 μL nuclease free (NF) water (NF water volume adjusted to yield atotal volume of 8 μL/reaction). The tube(s) comprising the hybridizationmixture was mixed gently and placed in a thermal cycler programmed for65° C. for ten minutes, then 16° C. for five minutes. The tube(s) werekept at 16° C. and ligation reaction compositions were prepared bycombining in order 8 μL of the thermal cycled hybridization mixture, 10μL 2× ligation buffer (100 mM Tris, 20 mM MgCl₂, 20 mM DTT, 2 mM ATP,40% PEG 8000, pH 7.0) and 2 μL ligase mix (10 U/μL bacteriophage T4 RNAligase 2 (Rnl2), 2 U/μL RNase Inhibitor Protein) for a final volume of20 μL for each reaction. The tube(s) were mixed by pipetting up and downand then incubated at 16° C. for 16 hours in the thermal cycler.Typically, 2 hour incubation is sufficient for a first adaptor and asecond adaptor to anneal with an RNA molecule and for ligation to occur,generating ligated product (see, e.g., 34 in FIG. 3).

The tube(s) comprising ligated product was placed on ice and a reversetranscription master mix (RTMM) prepared as follows. For each tube ofligated product, 134 NF water, 4 μL 10×RT buffer (500 mM Tris-HCl pH8.3, 750 mM KCl, 30 mM MgCl₂, 50 mM DTT, final pH 8.25), 2 μL 2.5 mMdNTP mix, and 1 μL RT enzyme mix (200 U/μL ArrayScript™ reversetranscriptase (RNA-directed DNA polymerase), were combined to form theRTMM. Twenty μL of RTMM was added to each ligated product tube withmixing by pipetting up and down 3-5 times followed by incubation at 42°C. for thirty minutes to generate reverse transcribed product (see,e.g., 36 in FIG. 3). After this incubation, a ten μL aliquot of thesolution comprising the reverse transcribed product was transferred to afresh 0.2 mL tube to which 1 μL of RNase H enzyme (10 U/μL E. coli RNaseH) was added. The tube(s) was mixed gently and incubated at 37° C. forthirty minutes, during which time the reverse transcribed product wasdigested and amplification template (see, e.g., 38 in FIG. 3) formed.

A PCR master mix was prepared comprising 38.5 μL NF water, 5 μL GeneAmp®PCR buffer I, 1 μL PCR primer mix (25 μM forward primer, 25 μM reversebarcoded primer), 4 μL 2.5 mM dNTP mix, and 1 μL AmpliTaq® DNApolymerase (5 U/μL; DNA-directed DNA polymerase)—a total of 49.5 μL peramplification reaction to be performed. This exemplary PCR primer mixincludes one forward primer and a barcoded reverse primer.

To determine the appropriate number of PCR cycles to use with a givensolution comprising amplified product, a small scale (i.e., 50 μL) PCRreaction is recommended. Small scale amplification reaction compositionswere prepared by combining 49.5 μL of this PCR master mix with 0.5 μL ofthe solution comprising amplification template in a RNase-free 0.2 mLthin-walled PCR tube. The amplification reaction compositions wereplaced in a thermal cycler with a heated lid, heated at 95° C. for fiveminutes, then cycled for 12-15 cycles using a profile of 95° C. for 30seconds-62° C. for 30 seconds-72° C. for 30 seconds; then a finalextension step is performed at 72° C. for 7 minutes. The optimum numberof amplification cycles depends on the amount of amplification productin the initial amplification reaction composition. A 5-10 μL aliquot ofthe amplification reaction composition comprising amplified product wasanalyzed by electrophoresis using a 6% native tris-borate EDTA (TBE)acrylamide gel to determine the optimum number of amplification cycles.Following such determination, a large scale amplification is performed.

To generate sufficient quantities of amplified product for sequencingand/or other downstream processes, a larger scale amplification reactionwas performed by PCR. Master mix was prepared by combining, for eachreaction to be performed, 77 μL NF water (Ambion AM9922), 10× GeneAmp®PCR Buffer I (or 10×PCR Buffer I, Applied Biosystems P/N N8080160), 2 μLbarcoded PCR primers (mix of forward and a barcoded reverse primer ofchoice), 8 μL dNTP mix, and 2 μL AmpliTaq® DNA-directed DNA Polymerase(Applied Biosystems P/N N8080160). A 99 μL aliquot of this master mixand 1 μL of the solution comprising amplification templates werecombined in three separate wells of an RNase-free PCR plate (i.e., intriplicate) to form an amplification reaction composition. The PCR platewas heated to 95° C. for 5 minutes to denature the nucleic acid, cycledaccording to the previous temperature profile (95° C. for 30 seconds-62°C. for 30 seconds-72° C. for 30 seconds) for the previously determinednumber of cycles and finally the PCR reaction vessel was maintained at72° C. for 7 minutes to generate amplified products in the amplificationreaction composition. The amplified products were pooled analyzed byelectrophoresing 5-10 μL of the pooled amplification reactioncompositions on a 6% native TBE acrylamide gel as before.

Two hundred and fifty (250) μL of the pooled amplified product wascombined with 250 μL phenol/chloroform/isoamyl alcohol (25:24:1, pH 7.9)in an RNase-free 1.5 mL polypropylene microfuge tube and mixed byvortexing. The tube was centrifuged at 12,000 rpm for 5 minutes at roomtemperature using a benchtop centrifuge. The aqueous phase was measuredand transferred to a fresh RNase-free 1.5 ml polypropylene microfugetube and an equal volume of 7.5 M ammonium acetate added to the tubealong with 1/100 volume of glycogen (or GlycoBlue™ Co-precipitant(Ambion) and 0.7 volumes isopropanol. The contents of the tube are mixedthoroughly, incubated at room temperature for 5 minutes, and thencentrifuged at 12,000 rpm for 20 minutes at room temperature. Theresulting supernatant was removed and discarded and the pellet washedthree times with 1 mL of 70% (v/v) ethanol. The pellet was air dried,then resuspended in 18 μL nuclease-free water to which 2 μL of 10×native gel loading dye is added. Ten μL of this suspension was added toeach of two wells of a native TBE PAGE gel which contains a 10 basepair(bp) molecular weight ladder (Invitrogen 10821-015) as a marker in oneof the other wells. The amplified products were electrophoresed in thegel at ˜140 V until the dye front is about to elute off the bottom edgeof the gel (˜30 minutes for a 1.0 mm, 8 cm×8 cm gel). The nucleic acidbands in the gel were stained using SYBR® Gold (Invitrogen, Carlsbad,Calif.) following the manufacturer's instructions and illuminated usingan ultraviolet light source. Using a clean razor blade, the gel wassliced in the lanes containing amplified product to obtain the nucleicacid in the size range of approximately 100 to 150 bp. A distinct bandat 100 bp likely represents undesired byproducts and was not includedwith the slice excised from the gel. Likewise, nucleic acid larger thanabout 200 bp was also avoided when certain sequencing methods wereemployed, for example but not limited to, emulsion PCR sequencing usingthe SOLiD™ System (Applied Biosystems).

A hole was made in the bottom of an RNase-free 0.5 mL polypropylenemicrofuge tube using a 21 gauge needle and the excised gel piece istransferred to the tube. This 0.5 mL tube was then placed inside anRNase-free 1.5 mL polypropylene microfuge tube and centrifuged for 3 minat 12,000 rpm to shred the gel. The 0.5 mL tube is removed and discardedand the outer 1.5 mL tube containing the gel fragments is placed on ice.Two hundred (200) μL PAGE elution buffer (1.5 M ammonium acetate in 1×TEbuffer pH 7.0) was added to tube, which was then incubated at roomtemperature for 20 minutes. After this first incubation, the supernatantwas removed and transferred to a clean RNase-free 1.5 mL polypropylenemicrofuge tube and an additional 250 μl of PAGE elution buffer was addedto the first tube (containing the gel fragments) and incubated for anadditional 40 minutes at 37° C. Following the second incubation, thesecond supernatant was collected and added to the first. Residual gelpieces were removed form the pooled supernatants using a spin column(Ambion Cat#10065) and centrifugation, according to the manufacturer'sinstructions.

The resulting liquid was combined with an equal volume ofphenol/chloroform/isoamyl alcohol (25:24:1, pH 7.9) in an RNase-free 1.5mL polypropylene microfuge tube and mixed by vortexing. The tube wascentrifuged at 12,000 rpm for 5 minutes at room temperature using abenchtop centrifuge. The aqueous phase was measured and transferred to afresh RNase-free 1.5 ml polypropylene microfuge tube and an equal volumeof 7.5 M ammonium acetate was added to the tube along with 1/100 volumeof glycogen (AM9510, Ambion; or GlycoBlue™ Co-precipitant AM9515,Ambion) and 0.7 volumes isopropanol. The contents of the tube were mixedthoroughly, incubated at room temperature for 5 minutes, and thencentrifuged at 12,000 rpm for 20 minutes at room temperature. Theresulting supernatant was removed and discarded and the pellet washedthree times with 1 mL of 70% (v/v) ethanol. The pellet was air dried,then resuspended in 204 NF water. The DNA comprising the amplifiedproduct was quantitated by determining the A₂₆₀ with a spectrophotometeror by analyzing on a 6% native PAGE gel, as described above.

To determine the sequence of the amplified product, emulsion PCR (ePCR)was performed on an Applied Biosystems SOLiD™ System according to theUser Guide (Applied Biosystems, P/N4391578; the “User Guide”). Toevaluate what concentration of amplified product that gives the bestsequencing results in a full scale ePCR on the SOLiD™ System, fourseparate ePCR reactions were performed at amplified productconcentrations of 0.2 pg/μL, 0.4 pg/μL, 0.6 pg/μL, and 0.8 pg/μL,followed by a titration/QC run according to the manufacturer'sinstructions (see particularly, the User Guide, Chapters 3 and 4). Whenthe optimal amplified product concentration was determined, a “fullscale” ePCR reaction was performed (see Section 3.1, Chapter 3 of theUser Guide). By determining the sequence of at least part of theamplified product, one can directly or bioinformatically identify theRNA molecule from which that amplified product was derived, therebydetecting that RNA molecule. Those in the art will appreciate that suchsequence information may be used to identify novel RNA molecules,including without limitation, small RNA discovery; may be used toquantitate the amount of one detected RNA molecule species in thestarting sample relative to the amount another detected RNA species andsuch information may be useful for, among other things, expressionprofiling of mRNA, miRNA or other RNA molecules of interest.

Example 4 Evaluation of Adaptor Overhang Length

First and second adaptors with various overhang lengths were synthesizedand evaluated in an effort to maximize ligation efficiency whileminimizing adaptor complexity.

Exemplary first adaptors comprised first oligonucleotides, depicted as“T3” in FIG. 7B wherein the first oligonucleotides comprised a DNAsequence from bacteriophage T3 and two ribonucleotides at the 3′ end,and second oligonucleotides, depicted as “27 N” in FIG. 7B wherein thesecond oligonucleotides comprised a complementary deoxyribonucleotidesequence from bacteriophage T3 and an overhang of 4, 6 or 8 degeneratedeoxyribonucleotides “N” at the 5′ end. The upper strand (firstoligonucleotide) of an illustrative first adaptor comprised a 27nucleotide sequence from the bacteriophage T3 promoter5′-CUCGAGAAUUAACCCUCACUAAAGGGA-3′ (SEQ ID NO:7), shown as “T3” in FIG.7B. The lower strand (second oligonucleotide) comprised thecomplementary sequence of the upper strand with either 4, 6, or 8degenerate nucleotides (depicted as “N” for illustration purposes inFIG. 7B) on the 5′ end of the lower strand 5-(N)_(4,6,8)TCCCTTTAGTGAGGGTTAATTCTCGAG-3′ (SEQ ID NO:8 (where N=8); SEQ ID NO:8 lacking either2 or 4 5′-N's (i.e., where N is 6 or 4)), depicted as “27 N” forillustration purposes in FIG. 7B.

Exemplary second adaptors comprised third oligonucleotides, depicted as“T7” in FIG. 7B wherein the third oligonucleotides comprised a DNAsequence from bacteriophage T7, and fourth oligonucleotides, depicted as“N 28” in FIG. 7B wherein the fourth oligonucleotides comprised acomplementary deoxyribonucleotide sequence from bacteriophage T7 and anoverhang of 4, 6 or 8 degenerate deoxyribonucleotides “N” at its 3′ end.The upper strand (third oligonucleotide of an illustrative secondadaptor comprised a 28 nucleotide sequence from the bacteriophage T7promoter 5′-PO₄-TCCCTATAGTGAGTCGTATTACGAATTC-3′(SEQ ID NO:9) shown as“T7” in FIG. 7B which comprises a 5′ phosphate group (shown as PO₄). Thelower strand (fourth oligonucleotide) comprised the complementarysequence of the upper strand with either 4, 6, or 8 degeneratenucleotides (depicted as “N” for illustration purposes in FIG. 7B) onthe 3′ end of the lower strand5′-GAATTCGTAATACGACTCACTATAGGGA(N)_(4,6,8)-3′ (SEQ ID NO:10 (where N=8);SEQ ID NO:10 lacking either 2 or 4 3′-N's (i.e., where N is 6 or 4)),depicted as “N 28” for illustration purposes in FIG. 7B.

Fifty picomoles (pmol) of either the “T3” first adaptors (see FIG. 7B),the “T7” second adaptors (see FIG. 7B), or both the T3 first adaptorsand the T7 second adaptors (both with the same number of degeneratenucleotides) in 2 μL water was incubated with 3 μL 2× HybridizationBuffer (300 mM NaCl, 20 mM Tris pH8, 2 mM EDTA) at 95° C. for 3 minutes,then cooled to 22° C. When the temperature reached 22° C., 1 μL of a0.13 μM 5′ ³²P-labeled synthetic microRNA pool (mirVana™ miRNA ReferencePanel 9.0, a pool of approximately 500 synthetic miRNA sequences fromthe Sanger miRBase 9.0 database (microrna.sanger.ac.uk/sequences/) inequimolar concentration) was added to each reaction tube, the tubes wereincubated at 65° C. for 10 minutes, then cooled down to 22° C.

Reaction mixtures were then incubated at 16° C. for 30 minutes, then 14μL ligation enzyme mix (0.5 μL RNA ligase 2 (10 μM), 2 μL 10× Rnl2buffer (100 mM Tris pH7, 20 mM MgCl₂, 20 mM Dithiothreitol, 20 mM ATP),1 μL RNase Inhibitor (Ambion AM2682, 40 U/μL), 10 μL 40% PEG 8000(Sigma-Aldrich 202452) and 0.5 μL RNase-free water) was added to eachtube. The reaction mixtures were incubated for another 16 hours at 16°C. Twenty μL of Gel Loading Buffer II (Ambion AM8546G) was then added toeach reaction and heated at 95° C. for 5 minutes. The ³²P-labeledproducts were resolved by 10% denaturing PAGE and visualized byautoradiography. Control reactions lacking (i) both RNA ligase 2 andadaptors or (ii) adaptors only, are shown in the first two lanes of FIG.7A. Substantial amounts of the double ligation product (ligated product)were observed in the lanes of the gel corresponding to ligation reactionmixtures comprising both the first adaptors and the second adaptors(i.e., T3-4+T7-4; T3-6+T7-6; and T3-8+T7-8), as shown in FIG. 7A.Therefore, adaptor overhang lengths of 4, 6, and 8 nucleotides providefor detection of small RNA.

Example 5 Adaptor Structure

Different structures of 5′ (first) and 3′ (second) adaptors with6-nucleotide degenerate overhangs were tested which included dsDNA (withthe exception of two 3′ terminal RNA bases on the upper strand (firstoligonucleotide) of the 5′ (first) adaptor (e.g., T3r2:27 6N and T7:6 N28 in FIG. 8B (the numbers 27 and 28 refer to the length of the firstand third oligonucleotides, respectively, as in Example 4) and dsRNA•DNAhybrid (upper (first and third oligonucleotides are RNA)-lower (secondand fourth oligonucleotides are DNA) for both adaptors (e.g., rT3:27 6Nand rT7:6 N 28 in FIG. 8B).

In this illustrative embodiment of FIG. 8B, top first adaptor (rT3:276N) comprises an upper strand (first oligonucleotide) comprising the T3sequence (SEQ ID NO:7) wherein all of the nucleosides compriseribonucleosides, shown as “rT3,” annealed to the lower strand (secondoligonucleotide) comprising the complementary sequence and 6 degeneratenucleotides on the 5′ end of the lower strand (SEQ ID NO:8 where N is6), depicted as “27 6N” for illustration purposes; and the otherillustrative first adaptor (T3r2:27 6N) comprises an upper strandcomprising the T3 sequence wherein all of the nucleosides comprisedeoxyribonucleosides except for the two 3′-most nucleosides whichcomprise ribose (SEQ ID NO:7 having 2 3′-terminal ribonucleotides),shown as “T3r2” annealed to the same lower strand as the top firstadaptor (SEQ ID NO:8 where N is 6) depicted as 27 6N.

Again referring to FIG. 8B, a top illustrative second adaptor (rT7:6 N28) comprises an upper strand (third oligonucleotide) comprising the T7sequence (SEQ ID NO:9) wherein all of the nucleosides compriseribonucleosides and which sequence comprises a 5′ phosphate group,depicted as “rT7” for illustration purposes, annealed to the lowerstrand which comprises the complementary sequence with 6 degeneratenucleotides on the 5′ end of the lower strand (SEQ ID NO:10 where N is6), depicted as “6N 28” for illustration purposes. The otherillustrative second adaptor (T7:6 N 28) comprises an upper strandcomprising the T7 sequence (SEQ ID NO:9) wherein all of the nucleosidescomprise deoxyribonucleosides and which sequence comprises a 5′phosphate group, depicted as “T7” for illustration purposes, annealed tothe same lower strand as the top second adaptor (SEQ ID NO:10 where N is6), depicted as 6N 28.

Fifty pmol of either the first adaptors, the second adaptors, or boththe first adaptors and the second adaptors, in 2 μL water was incubatedwith 3 μL 2× Hybridization Buffer (300 mM NaCl, 20 mM Tris pH8, 2 mMEDTA) at 95° C. for 3 minutes and cooled down to 22° C. One (1) μL of a0.13 μM 5′ ³²P-labeled miRNA pool (mirVana™ miRNA Reference Panel ascited above) was added to each reaction, then incubated at 65° C. for 10minutes, and cooled down to 22° C.

Reaction mixtures were incubated at 16° C. for 30 minutes, then 14 μL ofligation enzyme mix (0.5 μL RNA ligase 2 (10 μM), 2 μL 10× Rnl2 buffer(100 mM Tris pH7, 20 mM MgCl₂, 20 mM Dithiothreitol, 20 mM ATP), 1 μLRNase Inhibitor (Ambion AM2682, 40 U/μL), 10 μL 40% PEG 8000(Sigma-Aldrich 202452) and 0.5 μL RNase-free water) was added to eachtube. The reaction mixtures were incubated at 16° C. for another 16hours. Gel Loading Buffer II (20 μL, Ambion AM8546G) was then added toeach reaction and heated at 95° C. for 5 minutes. The ³²P-labeledproducts were resolved by 10% denaturing PAGE and visualized byautoradiography. As shown in FIG. 8A, substantial amounts of ligatedproducts (indicated by arrow) are observed only when the ligationreaction mixtures comprised both first adaptors and second adaptors(T3r2-6+T7-6; and rT3-6+rT7-6), but more undesired reaction byproduct(indicated by *) is seen with the combined rT3-6+rT7-6 adaptors thanwith combined T3r2-6+T7-6 adaptors.

Example 6 Adaptor Combinations

Three different combinations of first adaptors and second adaptors weretested for double ligation efficiency. These combinations included firstadaptors and second adaptors with both DNA upper strands (i.e., firstand third oligonucleotides are DNA with the exception that the firstoligonucleotide has two 3′ ribonucleotides), both RNA upper strands(i.e., first and third oligonucleotides), or RNA upper strand on 5′(first) adaptor (i.e., first oligonucleotide) and DNA upper strand on 3′(second) adaptor (i.e., third oligonucleotide) (see FIG. 9C for aschematic of the latter adaptor structure embodiment). One (1) μL ofeach adaptor (50 μM each) was combined and incubated with 3 μL 2×Hybridization Buffer (300 mM NaCl, 20 mM Tris pH8, 2 mM EDTA) at 95° C.for 3 minutes and cooled down to 22° C. One (1) μL of a 0.13 μM 5′³²P-labeled synthetic miRNA pool (cited above) was added to each 5 μLreaction, the reactions were then incubated at 65° C. for 10 minutes andthen cooled down to 22° C.

Reaction mixtures were then incubated at 16° C. for 30 minutes before 14μL of the ligation enzyme mix was added to each tube. Ligation mix wasprepared by combining 0.5 μL RNA ligase 2 (10 μM), 2 μL 10× Rnl2 buffer(100 mM Tris pH7, 20 mM MgCl₂, 20 mM Dithiothreitol, 20 mM ATP), 1 μLRNase Inhibitor (Ambion AM2682, 40 U/μL), 10 μL 40% PEG 8000(Sigma-Aldrich 202452) and 0.5 μL RNase-free water. The reactionmixtures were incubated at 16° C. for another 16 hours. Twenty (20) μLof Gel Loading Buffer II (Ambion AM8546G) was then added to eachreaction and heated at 95° C. for 5 minutes. The ³²P-labeled productswere resolved by 10% denaturing PAGE and visualized by autoradiography.FIG. 9A and FIG. 9B depict the resulting electropherograms of ligatedproducts. Adaptors with either DNA or RNA upper strands are doublyligated to generate “ligated product” as depicted in FIG. 9A.

Different ratios of adaptors (upper vs. lower strand, i.e., first vs.second oligonucleotide and third vs. fourth oligonucleotide) with andwithout ribonuclease H(RNase H) digestion were evaluated to maximizedouble ligation products while minimizing undesired by-productsgenerated by direct ligation between 5′ (first) and 3′ (second) adaptors(“Ligated adaptors” in FIG. 10A). Adaptors with different upper/lowerstrand ratios as indicated in the table of FIG. 10A were mixed witheither 0.2 or 2 pmol mirVana™ miRNA Reference Panel (cited above) and 3μL 2× Hybridization Buffer (300 mM NaCl, 20 mM Tris pH8, 2 mM EDTA) in a6 μL reaction mixture followed by incubating at 65° C. for 15 minutesand 16° C. for 45 minutes. Ligation mix was prepared by combining 1 μLRNA ligase 2 (10 μM), 1 μL RNase Inhibitor (Ambion AM2682, 40 U/μL), 2μL 10× Rnl2 buffer (100 mM Tris pH7, 20 mM MgCl₂, 20 mM Dithiothreitol,20 mM ATP) and 10 μL 40% PEG 8000 (Sigma-Aldrich 202452). Ligation mix(14 μL) was added to each tube and incubated at 16° C. for 16 hours.

After 16 hours' incubation, 4 μL 10×RT Buffer which contains 500 mMTris-HCl (pH8.3), 750 mM KCl, 30 mM MgCl₂ and 100 mM DTT was combinedwith 2 μL 2.5 mM dNTP, 1 μL ArrayScript™ reverse transcriptase (200U/μL, Ambion AM2048), 1 μL SUPERase•In™ RNase inhibitor (20 U/μL, AmbionAM2694), and 12 μL RNase-free water and then added to 20 μL of ligationreaction for a total of 40 μL of RT mixture. The reverse transcription(RT) reaction mixture was incubated at 42° C. for 30 minutes.

For the samples of lanes 1 to 6 shown on the gel in FIG. 10A, PCR wasperformed as follows. One (1) μL sample from the RT mixture was combinedwith 10 μL 10× Complete Buffer (Ambion AM2050), 1 μL dNTP (25 mM), 0.5μL 5′ primer and 0.5 μL 3′ primer (50 μM each), 1 μL of SuperTaq DNApolymerase (5 U/μL, Ambion AM2050) and 86 μL of RNase-free water. PCRwas performed using 20 cycles of 30 seconds at 95° C., 30 seconds at 50°C. and 30 seconds at 72° C. Five (5) μL of PCR products were loaded on a10% native PAGE gel and visualized by SYBR® Gold (Invitrogen 11494)staining.

For the samples of lanes 7-12 shown on the gel in FIG. 10A, an RNase Hdigestion reaction was performed as follows followed by PCR. Five μL ofthe RT reaction was transferred to a clean tube, mixed with 0.5 μLRibonuclease H(RNase H, 10 U/μL, Ambion AM2292) and incubated at 37° C.for 30 minutes. One (1) μL of RNase H treated sample was used for PCRreaction at the same condition as described previously. All of theseamplified products (both with and without RNase H digestion) were loadedon a 10% native PAGE gel, electrophoresed, and visualized by SYBR® Gold(Invitrogen 11494) staining, as shown in FIG. 10A.

Adaptors with different upper/lower strand ratios as indicated in thetable shown in FIG. 10B were mixed with 2 pmol synthetic mirVana™ miRNAReference Panel (cited above) and 3 μL 2× Hybridization Buffer (300 mMNaCl, 20 mM Tris pH8, 2 mM EDTA) in a 6 μL reaction mixture followed byincubating at 65° C. for 15 minutes and 16° C. for 1 hour. The ligation,RT and RNase H treatment were performed as described previously. One (1)μL of RNase H treated sample was used for PCR amplification withSuperTaq™ polymerase (Ambion AM2050) in 20 cycles as described before.Five μL of each of the PCR products were loaded on a 10% native PAGEgel, electrophoresed, and visualized by SYBR® Gold (Invitrogen 11494)staining, as shown in FIG. 10B. Adaptors with picomolar ratios of upperto lower strand of 1/50, 5/50 10/50, 25/50 and 5/100 were all competentto generate the desired products migrating at >50 bp. In contrast, useof an adaptor ratio of 5/500 did not efficiently generate the desiredproducts.

Example 7 Comparison of the Present Method with TaqMan® miRNA AssaysSamples that Vary in RNA Content

The present example provides a quantitative validation of the presentmethods as compared to the RT-PCR TaqMan® miRNA assays. FIG. 12 depictsa scatter plot depicting a relative fold change (FC) comparison of miRNAquantitation results obtained from human placental RNA and from humanlung RNA using 5′ nuclease assays with sequencing results obtainedaccording to certain embodiments of the current teachings. The x-axisshows the log₂ fold change of 5′ nuclease assay results in −ΔΔCT((generated using TaqMan® Human MicroRNA Array v1.0 (P/N 4384792;Applied Biosystems) and Multiplex RT for TaqMan® MicroRNA Assays (P/Ns4383403, 4383402, 4383401, 4383399, 4384791, 4382898, 4383405, 4383400,and 4383404; Applied Biosystems) performed essentially according to themanufacturer's protocol) and the y-axis shows the log₂ fold change ofsequencing data (generated using the SOLiD™ Sequencing System (AppliedBiosystems) essentially according to the manufacturer's protocol)according to an embodiment of the current teachings. TaqMan® MicroRNAAssays (Applied Biosystems) that generated C_(T) values above 35 werepresumed to be negative and not included in the analysis. For SOLiD™sequencing data, data from at least 3 sequenced tags were required forthe corresponding sequence to be considered as ‘observed.’ Using thisapproach an R value of 0.88 was obtained.

The present example also provides for total RNA input amounts since somesample types contain minimal small RNAs. One embodiment of a currentmethod was performed using total RNA from either human placenta (FIG.13A) or mouse liver (FIG. 13B) at the amounts indicated in the figures.In general, adaptor mix A (2 μl) (SOLiD™ adaptor mix A) containing 25pmol RNA upper strand (first oligonucleotide) and 50 pmol DNA bottomstrand (second oligonucleotide) with 6 degenerate DNA nucleotidesforming an overhang at the 5′ end as 5′ (first) adaptor, and 25 pmol5′-phosphorylated DNA upper strand (third oligonucleotide) and 50 pmolDNA bottom strand (fourth oligonucleotide) with 6 degenerate DNAnucleotides forming an overhang at the 3′ end as 3′ (second) adaptorwere incubated with 3 μl 2× Hybridization Buffer (300 mM NaCl, 20 mMTris pH8, 2 mM EDTA) and 1 μl total RNA with indicated amounts at 65° C.for 10 minutes and 16° C. for 5 minutes.

A ligation mix was prepared by combining 1 μL RNA ligase 2 (10 μM), 1 μlRNase Inhibitor (Ambion AM2682, 40 U/μL), 2 μL 10× Rnl2 buffer (100 mMTris pH7, 20 mM MgCl₂, 20 mM Dithiothreitol, 20 mM ATP), and 10 μL 40%PEG 8000 (Sigma-Aldrich 202452). The ligation reaction mixtures wereincubated at 16° C. for 16 hours.

After 16 hours' incubation, 4 μL 10×RT Buffer which contains 500 mMTris-HCl (pH8.3), 750 mM KCl, 30 mM MgCl₂ and 100 mM DTT was combinedwith 2 μL 2.5 mM dNTP, 1 μL ArrayScript™ reverse transcriptase (200U/μL, Ambion AM2048), and 13 μL RNase-free water and then added to 20 μLligation reaction composition. The reverse transcription (RT) reactionmixture was incubated at 42° C. for 30 minutes.

Then 5 μL of RT reaction was transferred to a clean tube, mixed with 0.5μL Ribonuclease H(RNase H, 10 U/μL, Ambion AM2292) and incubated at 37°C. for 30 minutes. 0.5 μL of RNase H treated sample was then combinedwith 5 μL 10× AmpliTaq® Buffer I (Applied Biosystems N8080171), 4 μLdNTP (2.5 mM), 0.5 μL 5′ primer and 0.5 μL 3′ primer (50 μM each), 1 μLof AmpliTaq® DNA polymerase (5 U/μL, Applied Biosystems N8080171) and38.5 μL of RNase-free water. PCR was performed using 16 cycles of 30seconds at 95° C., 30 seconds at 62° C. and 30 seconds at 72° C. Five(5) μL of PCR products were loaded on a 10% native PAGE gel andvisualized by SYBR® Gold (Invitrogen 11494) staining. As shown by FIG.13A, note that the placenta sample is fairly rich in small RNAs and themethod presented herein is capable of producing small RNA products from≦25 ng total RNA. In contrast, the mouse liver sample as shown by FIG.13B is very poor in small RNAs by comparison and thus, according tocertain embodiments of the current teachings, enrichment of such samplesmay provide better results.

Sequences for the adaptor Mix A are as for SEQ ID NO:1-SEQ ID NO:4 ofExample 1.

Example 8 Real-Time PCR Demonstrates Dynamic Range of Small RNA LibraryConstruction

Four synthetic RNA oligonucleotides containing 5′-PO4 (spike incontrols, SIC; sequences shown below) were mixed at varyingconcentration spanning a 1000-fold input range (1000, 100, 10, and 1 pgin 1000× mix), and the mixture was serially diluted to 1×. Mixtures werespiked into 500 ng placenta total RNA as background (FIG. 14, FIG. 16A)and the ligation reaction was performed on the four samples as describedpreviously except that the ligation reaction composition was incubatedfor two hours (instead of sixteen hours) at 16° C.

The samples were then treated with RNase H as described above and 0.5 μLof RNase H treated sample was then combined with 5 μL 10× AmpliTaq®Buffer I (Applied Biosystems N8080171), 4 μL dNTP (2.5 mM), 0.5 μL 5′primer and 0.5 μL 3′ primer (50 μM each), 1 μL of AmpliTaq® DNApolymerase (5 U/μL, Applied Biosystems N8080171) and 38.5 μL ofRNase-free water. PCR was performed using 15 cycles of 30 seconds at 95°C., 30 seconds at 62° C. and 30 seconds at 72° C. Following PCR cleanupby Qiagen PCR purification kit (Qiagen 28104), samples were eluted in 30μL water and further diluted in a 1:400 ratio. Two μL diluted sample wasthen combined with 12.5 μL 2×SYBR® Green PCR Master Mix (AppliedBiosystems 4309155), 1 ul primer mix (1 μM each) and 9.5 μL water(schematically shown in FIG. 15). Real-Time PCR was performed on an ABI7500 Real-Time PCR System (Applied Biosystems 4351104) using 95° C. for10 minutes, 40 cycles of 15 seconds at 95° C. and 1 minute at 68° C.,followed by 15 seconds at 95° C., 1 minutes at 60° C. and 15 seconds at95° C.

Cycle threshold values (Cts) for each target sequence (FIG. 16A, y-axis:Mean Ct) were plotted against log 10 values representing the four sampleinputs y-axis: log(mix concentration) 1000× to 1×, FIG. 16A). As seen inFIG. 16B, Cts for two endogenous miRNA control sequences in thebackground RNA (miR-16 and miR-21) were fairly constant across samples.

The four synthetic RNA oligonucleotides used in this example as “spikein controls” (SIC) were: (1) SIC 8: 5′-Phos GCGAAUUAAUGAAAGUGGGCA (SEQID NO:11; data shown using triangles in FIG. 16A); (2) SIC 34: 5′-PhosACCCGACAUUAAAGGUGGCAU (SEQ ID NO:12; data shown using circles in FIG.16A); (3) SIC 36: 5′-Phos CUCACAUUUCGGAACUGAUGC (SEQ ID NO:13; datashown using diamonds in FIG. 16A); and (4) SIC 37: 5′-PhosACGGACCUCGAACUUCACCCA (SEQ ID NO:14; data shown using squares in FIG.16A). Therefore, the spike-in-control assays demonstrate an ability ofthe present methods to detect small RNA in a dynamic range spanning atleast 1000-fold.

Example 9 One-Step RT-PCR

The present example provides an exemplary method for generatingamplification templates and amplified products, wherein the RNA-directedDNA Polymerase and DNA-directed DNA Polymerase are in the ReverseTranscription Reaction Composition.

Following the ligation reaction a mixture containing appropriate buffer,dNTPs, ArrayScript™ reverse transcriptase, AmpliTaq® DNA polymerase andboth forward and reverse PCR primers are added directly to the ligationreaction and mixed by gentle pipetting up and down several times (3-4×).The reaction mixture is then incubated at 42° C. for 30 minutes in athermal cycler to permit reverse transcription to generate anamplification template. RNase H is then added to the reaction mixtureand incubated at 37° C. for 30 minutes in a thermal cycler. The reactiontemperature is then ramped up to 95° C. and held for 5 minutes followedby standard amplification cycles, i.e., 30 seconds at 62° C. and 30seconds at 72° C., as described in Example 1 to generate amplifiedproducts.

Example 10 Generating Amplification Templates and Amplified Productswith One DNA Polymerase

Following the ligation reaction a mixture containing appropriate buffer(containing optimized concentrations of both manganese and magnesium),dNTPs, a DNA polymerase comprising both DNA dependent DNA polymeraseactivity and RNA dependent DNA polymerase activity and both forward andreverse PCR primers is added to the ligation reaction composition andmixed by gentle pipetting up and down several times (3-4×) to from areverse transcription composition. The reverse transcription compositionis incubated at 42° C. for 30 minutes in a thermal cycler to permit thereverse transcriptase activity to generate an amplification template.RNase H is then added to the reaction mixture and incubated at 37° C.for 30 minutes in a thermal cycler. The reaction temperature is thenramped up to 95° C. and held for 5 minutes followed by standardamplification cycles, i.e., 30 seconds at 62° C. and 30 seconds at 72°C., as described in Example 1 to generate amplified products.

Example 11 Exemplary Method for Generating a Small RNA Library

The present example provides exemplary methods for generating a smallRNA library as depicted in FIG. 17. When the RNA molecule of interest isa small RNA, the starting material should comprise the small RNAfraction. FirstChoice® prepared Total RNA (Applied Biosystems) iscertified to contain miRNA and other small RNAs. Alternatively, totalRNA that includes the small RNA fraction of a sample may be obtainedusing the mirVana™ miRNA Isolation Kit or mirVana™ PARIS™ Kit accordingto user's manual following the procedures for total RNA isolation.

Since RNA samples can vary widely in small RNA content based on theirsource and the RNA isolation method, evaluating the small RNA content ofsamples to determine whether to use total RNA or size-selected RNA inreactions may be desirable, using for example, but not limited to, anAgilent bioanalyzer with the Small RNA Chip.

Total RNA samples that contain more than 0.5% small RNA (in the ˜10-40nt size range) can be used without size-selection. When total RNA isused in the procedure the resulting reaction products will be a largersize range than those produced from PAGE-purified small RNA samples. Inaddition, SOLiD™ sequencing results from total RNA samples willtypically include a slightly higher number of rRNA and tRNA reads.

RNA samples that contain less than 0.5% small RNA content should beenriched for the ˜18-40 nt RNA fraction, for example but not limited to,by PAGE and elution, by the flashPAGE™ Fractionator and flashPAGE™Reaction Clean-Up Kit (Applied Biosystems).

The relative amount of small RNA in different sample types variesgreatly as described in Example 7. For example, RNA from tissue samplestypically has a rich supply of small RNAs, whereas RNA from culturedcell lines often has very few small RNAs.

Recommended input RNA quantities include:

RNA Source Amount Total RNA isolated from tissue 10-500 ng Total RNAisolated from cultured cells 100-500 ng Small RNA size-selected usingPAGE 1-200 ng Control RNA (human placenta total RNA) 100 ng

Hybridization and Ligation:

Hybridization and Ligation are carried out as follows. An adaptor mix Ais designed for SOLiD™ sequencing from the 5′ ends of small RNAs, forexample, and an adaptor mix B is designed for SOLiD™ sequencing from the3′ ends. To sequence the small RNA in a sample from both the 5′ and 3′ends, two ligations were set up, one with each adaptor mix. Each adaptormix contains first, second, third and fourth oligonucleotides. Adaptormix B is in the reverse complement orientation as compared to adaptormix A so that each strand of an amplified product can be detected. Onice, the hybridization mix is prepared in 0.2 mL PCR tubes as follows.

Hybridization Mixture (8 μL total volume) Amount Component 2 μL AdaptorMix A or B 3 μL Hybridization Solution 1-3 μL RNA sample (1-500 ng) to 8μL Nuclease-free Water

The contents were mixed well by gently pipetting up and down a fewtimes, then centrifuged briefly to collect the solution at the bottom ofthe tube. The reactions were placed in a thermal cycler with a heatedlid, programmed as follows.

Adaptor Hybridization Incubation Temperature Time 65° C. 10 min 16° C.hold

The sample was incubated at 16° C. for 5 minutes. Maintaining thereaction(s) at 16° C., add the RNA ligation reagents to each sample inthe order shown.

Ligation Reaction Mix (20 μL final volume) Amount Component (add inorder shown) 10 μL 2X Ligation Buffer 2 μL Ligation Enzyme Mix

The mix was incubated for 16 hours in a thermal cycler set to 16° C. A 2hour incubation is generally sufficient for ligation, however, anovernight incubation resulted in slightly higher amounts of ligatedproduct.

Reverse Transcription and RNase H Digestion:

The sample(s) were placed on ice and a Reverse Transcription (RT) MasterMix was prepared on ice by combining the following reagents. An extra5-10% volume was included in the master mix to compensate for pipettingerrors.

RT Master Mix (20 μL per sample) Amount Component 13 μL Nuclease-freeWater 4 μL 10X RT Buffe 2 μL dNTPs 1 μL ArrayScript ReverseTranscriptase 20 μL Total volume per reaction

RT Master Mix (20 μL) was added to each sample and the samples werevortexed gently to mix thoroughly and microcentrifuged briefly tocollect the mixture at the bottom of the tube. The samples were thenincubated at 42° C. for 30 minutes to synthesize cDNA.

The cDNA can be stored at −20 C for a few weeks, at −80° C. for longterm storage, or used immediately in the RNase H digestion (next).

RNase H incubation was carried out as follows. A volume (10 μL) of theRT reaction mixture was transferred from the previous step (cDNA) to afresh tube. RNase H (1 μL) was added. The mixture was vortexed gently tomix, microcentrifuged briefly to collect the mixture at the bottom ofthe tube, and incubated at 37° C. for 30 minutes.

After the RNase H treatment, samples can be stored at −20° C. overnightor used immediately in the PCR.

Small RNA Library Amplification:

Pilot and Large Scale PCRs: Because different sample types can containsubstantially different amounts of small RNA, the number of PCR cyclesneeded to obtain enough DNA for SOLiD™ sequencing also varies. A 50 μLtrial PCR was performed to determine the number of PCR cycles needed fora given sample type before proceeding to a set of three or morereplicate 100 μL reactions (Large Scale PCRs) used to synthesizetemplate for the next step in SOLiD™ sequencing sample preparation.

Most samples should be amplified for 12-15 cycles. For pilotexperiments, 12 PCR cycles are recommended for samples from startingmaterial with a relatively high amount of small RNA (i.e., total RNAfrom tissue of ˜200-500 ng, or ˜50-200 ng size-selected small RNA) and15 cycles for those with relatively little small RNA (i.e., total RNAfrom tissue of ˜1-200 ng, or total RNA from cultured cells of ˜100-500ng, or 1-50 ng size-selected small RNA.

Small RNA PCR Primer Sets:

Ten different PCR primer sets for synthesis of SOLiD™ sequencingtemplate are provided with the SOLiD™ Small RNA Expression Kit (AppliedBiosystems). The primer sets are identical except for a 6 bp barcodelocated near the middle of the primers. This barcode feature of the PCRprimers enables sequencing and analysis of multiplexed samples. That is,it is possible to sequence up to ten different samples, one amplifiedwith each of the supplied SOLiD™ Small RNA PCR Primer Sets, in a singleSOLiD™ sequencing reaction. Any of the primer sets can be used butsamples are not mixed at this point.

Exemplary PCR primers include without limitation the forward primer andbarcoded reverse primers shown below (individual barcode sequences areunderlined).

Forward Primer (SEQ ID NO: 5):5′-CCACTACGCCTCCGCTTTCCTCTCTATGGGCAGTCGGTGAT-3′Reverse Primer BC1 (SEQ ID NO: 15):5′-CTGCCCCGGGTTCCTCATTCTCTAAGCCCCTGCTGTACGGCCAA GGCG-3′Reverse Primer BC2 (SEQ ID NO: 16):5′-CTGCCCCGGGTTCCTCATTCTCTCACACCCTGCTGTACGGCCAA GGCG-3′Reverse Primer BC3 (SEQ ID NO: 17):5′-CTGCCCCGGGTTCCTCATTCTCTCCCCTTCTGCTGTACGGCCAA GGCG-3′Reverse Primer BC4 (SEQ ID NO: 18):5′-CTGCCCCGGGTTCCTCATTCTCTCATCGGCTGCTGTACGGCCAA GGCG-3′Reverse Primer BC5 (SEQ ID NO: 19):5′-CTGCCCCGGGTTCCTCATTCTCTTCGTTGCTGCTGTACGGCCAA GGCG-3′Reverse Primer BC6 (SEQ ID NO: 20):5′-CTGCCCCGGGTTCCTCATTCTCTGGGCACCTGCTGTACGGCCAA GGCG-3′Reverse Primer BC7 (SEQ ID NO: 6):5′-CTGCCCCGGGTTCCTCATTCTCTCCAGACCTGCTGTACGGCCAA GGCG-3′Reverse Primer BC8 (SEQ ID NO: 21):5′-CTGCCCCGGGTTCCTCATTCTCTCTCCGTCTGCTGTACGGCCAA GGCG-3′Reverse Primer BC9 (SEQ ID NO: 22):5′-CTGCCCCGGGTTCCTCATTCTCTCCCTTCCTGCTGTACGGCCAA GGCG-3′Reverse Primer BC10 (SEQ ID NO: 23):5′-CTGCCCCGGGTTCCTCATTCTCTGCGGTCCTGCTGTACGGCCAA GGCG-3′

A PCR Master Mix was prepared on ice by combining reagents as followsfor a single 50 μL Trial PCR or a 100 μL Large Scale PCR.

PCR Master Mix (for a Single Reaction) Trial PCR Large Scale PCR (50 μL)(100 μL) Component 38.9 μL 77.8 μL Nuclease-free Water 5 μL 10 μL 10XPCR Buffer I 1 μL 2 μL SOLiD Small RNA PCR Primer Set (one set) 4 μL 8μL 2.5 mM dNTP Mix 0.6 μL 1.2 μL AmpliTaq ® DNA Polymerase 49.5 μL 99 μLTotal volume per reaction

The mix was vortexed gently to mix thoroughly and microcentrifugedbriefly to collect the mixture at the bottom of the tube. (Once theappropriate number of PCR cycles for the sample type was determined, 3or more replicate large scale PCRs are run for each sample. Reactionproducts are pooled to generate enough material for gel purification andsubsequent SOLiD™ sequencing sample preparation.)

PCR Master Mix for a single reaction was pipetted into wells of a PCRplate or 0.2 mL PCR tubes. For a trial PCR (50 μL), 0.5 μL RNaseH-treated cDNA was added to each aliquot of PCR Master Mix. For largescale PCR (100 μL), 1 μL RNase H-treated cDNA was added to each aliquotof PCR Master Mix. Greater than 1 μL cDNA in a 50 μL PCR is notrecommended due to possible reaction inhibition.

Sample(s) were placed in a thermal cycler with a heated lid and thethermal profile shown below was carried out.

PCR Cycling Conditions Stage Reps Temp Time Denaturation (hold) 1 1 95°C. 5 min PCR (cycle) 2 12-15 95° C. 30 sec 62° C. 30 sec 72° C. 30 secFinal Extension 3 1 72° C. 7 min

PCR product (5-10 μL) was run on a native 6% polyacrylamide gel and thegel is stained with SYBR® Gold following the manufacturer'sinstructions.

FIG. 5 shows results from reactions that were amplified using anappropriate number of PCR cycles. Results are discussed in Example 1 andgeneralized here for illustration.

The amplified product derived from small RNA migrates at ˜108-130 bp(the total length of the primers is ˜89 bp, therefore, RNA of about19-41 bp migrate in the cited range).

Note that higher molecular weight bands at approximately 150 and 200 bpare expected from reactions using total RNA as input, whereas theselarger products are not expected from reactions using size-selectedsmall RNA as input (see FIG. 5).

Self-ligated adaptors and their amplified products form a band at 89 bp.This band is typically present in all reactions. Further by-productsmigrate at ˜100 bp. Underamplified samples exhibit very little materialin the ˜108-130 bp size range. Conversely, overamplified samplestypically show a significant amount of material in the ˜108-130 bp sizerange, plus a smear of reaction products larger than ˜140-150 bp.Overamplified samples from total RNA input may also have a highermolecular weight ladder of bands that represent concatenated PCRproducts.

Amplified Small RNA Library Cleanup:

The PCR products derived from small RNA were then cut from the gel,eluted out of the acrylamide, purified, and concentrated as follows.Samples were not heated at any step of this purification so that the DNAduplexes remain annealed and migrate according to their size duringsubsequent gel purification.

An equal volume of phenol/chloroform/isoamyl alcohol (25:24:1, pH 7.9)was added to each sample. Samples were vortexed to mix, then centrifugedat 13,000×g for 5 min at room temperature. The aqueous (upper) phase wastransferred to a fresh 1.5 mL tube, with the volume measured duringtransfer. An equal volume of 5 M ammonium acetate was added to eachsample. An amount (1/100 volume) of glycogen and 0.7 volume isopropanolwere added (the sample volume after addition of ammonium acetate is usedas a baseline). The mixture was mixed thoroughly, incubated at roomtemperature for 5 minutes, and centrifuged at 13,000×g for 20 minutes atroom temperature. The supernatant was carefully removed and discarded.The DNA pellet was washed 3 times with 1 mL of 70% ethanol each time andallowed to air dry for ˜15 minutes or until visible droplets of ethanolhad evaporated.

PAGE gels were prepared, e.g., 0.75 mm, native TBE, 6% polyacrylamidegel. The size of the gel is not important; minigels (˜60-100 cm2) aretypically the most convenient. Gels cast in-house within a few hours ofuse provide better resolution than purchased pre-cast gels. PAGE elutionbuffer is also prepared. For example, for 10 mL elution buffer, 5 mL ofTE Buffer pH 8 (10 mM Tris-HCl, pH 8, 1 mM EDTA) is combined with 5 mLof 5 M Ammonium acetate (2.5 M final conc); ˜450 μL is needed for eachsample.

A needle (e.g., 21-gauge) was used to poke a hole through thebottom-center of 0.5 mL microcentrifuge tube for each sample. The gelpieces excised above were placed in these tubes, and the centrifugationin the subsequent step shred the DNA-containing gel pieces for elutionof the DNA. The DNA pellet from above in 20 μL 1× nondenaturing gelloading buffer was loaded onto a 6% native TBE polyacrylamide gel. A DNALadder, or a similar ladder, was loaded in a separate lane as a marker.The gel was run at ˜140 V (˜30 minutes for a minigel) or until theleading dye front almost exits the gel and stained with SYBR® Goldfollowing the manufacturer's instructions. The gel piece containing105-150 bp DNA was excised using a clean razor blade and placed in a 0.5mL tube prepared with a hole in the bottom, the 0.5 mL tube placedwithin a larger 1.5 mL tube. The gel piece was shredded bymicrocentrifuging for 3 min at 13,000×g. An amount (200 μL) PAGE elutionbuffer was added to the shredded gel pieces; the mixture incubated atroom temperature for 1 hour, and the buffer was transferred to a freshtube, leaving the gel fragments behind. The shredded gel pieces wereextracted again, this time at 37° C. and the elution buffers werecombined. Transfer the mixture to a Spin Column and centrifuged at topspeed for 5 minutes to remove gel pieces. The DNA was in theflow-through.

An amount (1/100 volume) of glycogen and 0.7 volume isopropanol wereadded to each sample. The samples were mixed thoroughly, incubated atroom temperature for 5 minutes, and centrifuged at 13,000×g for 20 minat room temperature. The supernatant was carefully removed and discardedand the pellet was air dried, then resuspended in 20 μL nuclease-freewater.

DNA in each sample was quantitated by measuring the A₂₆₀ in aspectrophotometer (1 A₂₆₀=50 μg DNA/mL) and verifying the size andquality using an Agilent bioanalyzer or 6% native PAGE. The minimumamount of DNA that can be used for SOLiD™ sequencing is 200 ng at 20ng/L, but more DNA is preferable. SOLiD™ Small RNA Expression Kitreaction products enter the SOLiD™ sample preparation workflow at the“SOLiD™ System Template Bead Preparation” stage, in which emulsion PCRis used to attach molecules to beads. SOLiD™ sequencing and emulsion PCRis described, for example, in published PCT applications WO 06/084132entitled “Reagents, Methods, and Libraries For Bead-Based Sequencing andWO 07/121,489 entitled “Reagents, Methods, and Libraries for Gel-FreeBead-Based Sequencing.”

Example 12 Validation of Present Methods for Mapping and TranscriptCoverage

The present example provides for validation of present methods bydemonstrating that the methods are useful for mapping short (25-70 base)tags of sequences to the human genome or other databases and providesuniform transcript coverage.

For example, at least 15 libraries testing different conditions wereseparately made, pooled and then sequenced in a single run. A relativelyequal number of miRNA sequences were found across the libraries; anydeviations from equal numbers detected likely represent pipetting errorsin the pooling step. In a single sample, 389 out of 555 miRNAs weredetected (70%). Undetected miRNAs predominantly represent RNA moleculesthat are not present in the sample.

Sequence reads cs.fasta files generated on the SOLiD™ instrument weremapped to databases of miRNAs, tRNAs, rRNAs, Refseqs and the genome. Outof 17.7 million reads, 55% were mapped to the genome, 12% to RefSeqsequences, 1% to rRNA, 0.5% to tRNA, 8.25% to miRNA and 23% were notmapped. Reads that mapped to the genome uniquely were found to beclustered, i.e., ˜3000 have at least two reads and a size of <70 bases.These sequences are candidates for novel miRNAs or ncRNAs.

An analysis of transcriptome sequencing using the SOLiD™ System offifteen barcoded and pooled libraries found that, out of about 59.16million reads, 36.22% were mapped to the genome, 6.98% to RefSeqsequences, 0.32% to rRNA, and 0.01% to tRNA.

RNA detection was demonstrated to be reproducible. Placenta poly(A)RNase III fragmented libraries generated under similar but not identicalconditions were found to have good reproducibility (R2 values of 0.97)and a dynamic range of detection spanning at least 5 logs.

A whole transcript analysis was carried out using the SOLiD™ System andthe coverage for mRNA to brain-specific angiogenesis inhibitor 2 (BAI2)was found to be uniform from 5′ to 3′. Overlapping individual 50 basetags representing nearly the entire length of the mRNA were observed.Specific tags represented multiple times can be a reflection of therelative amount of the mRNA contained within the sample (that is, themore RNA molecules present in the sample the higher the probability ofcapturing the same tag of sequence), bias in the cleavage site of thefragmented RNA, or possibly PCR amplification artifacts.

The density of starting points for sequencing tags was plotted relativeto the length of the RNA transcripts analyzed. The results indicatedthat tag density is uniform across the body of the transcripts and thereis a drop off at the extreme 5′ and 3′ ends. This decrease in capturetags at the ends of the RNA represent the inefficiency of ligating tothe 5′ cap contained on all RNA polymerase II generated RNA transcripts.Regarding the 3′ end of mRNAs that typically contain a polyA tail, theability to capture and amplify this material appears hindered and theproportion of tags is low, possibly due to the homopolymer nature of thetail.

The compositions, methods, and kits of the current teachings have beendescribed broadly and generically herein. Each of the narrower speciesand sub-generic groupings falling within the generic disclosure alsoform part of the current teachings. This includes the genericdescription of the current teachings with a proviso or negativelimitation removing any subject matter from the genus, regardless ofwhether or not the excised material is specifically recited herein.

Although the disclosed teachings have been described with reference tovarious applications, methods, and compositions, it will be appreciatedthat various changes and modifications may be made without departingfrom the teachings herein. The foregoing examples are provided to betterillustrate the present teachings and are not intended to limit the scopeof the teachings herein. Certain aspects of the present teachings may befurther understood in light of the following claims.

1. A method for detecting a RNA molecule in a sample, comprising:combining the sample with at least one first adaptor, at least onesecond adaptor, and a polypeptide comprising double-strand specific RNAligase activity to form a ligation reaction composition in which the atleast one first adaptor and the at least one second adaptor are ligatedto the RNA molecule of the sample to form a ligated product in the sameligation reaction composition, wherein the at least one first adaptorcomprises: a first oligonucleotide having a length of 10 to 60nucleotides and comprising at least two ribonucleosides on the 3′-end,and a second oligonucleotide comprising a nucleotide sequencesubstantially complementary to the first oligonucleotide and furthercomprising a single-stranded 5′ portion of 1 to 8 nucleotides when thefirst oligonucleotide and the second oligonucleotide are duplexed,wherein the at least one second adaptor comprises: a thirdoligonucleotide having a length of 10 to 60 nucleotides and comprising a5′ phosphate group, and a fourth oligonucleotide comprising a nucleotidesequence substantially complementary to the third oligonucleotide andfurther comprising a single-stranded 3′ portion of 1 to 8 nucleotideswhen the third oligonucleotide and the fourth oligonucleotide areduplexed, wherein the single-stranded portions independently have adegenerate nucleotide sequence, or a sequence that is complementary to aportion of the RNA molecule, wherein the first and thirdoligonucleotides have a different nucleotide sequence; wherein the RNAmolecule hybridizes with the single-stranded portion of the at least onefirst adaptor and the single-stranded portion of the at least one secondadaptor; and detecting the RNA molecule of the ligated product or asurrogate thereof.
 2. The method of claim 1 wherein detecting the RNAmolecule or a surrogate thereof comprises: combining the ligated productwith i) a RNA-directed DNA polymerase, ii) a DNA polymerase comprisingDNA dependent DNA polymerase activity and RNA dependent DNA polymeraseactivity, or iii) a RNA-directed DNA polymerase and a DNA-directed DNApolymerase, reverse transcribing the ligated product to form a reversetranscribed product, combining the amplification template with at leastone forward primer, at least one reverse primer, and a DNA-directed DNApolymerase when the ligated product is combined as in i), to form anamplification reaction composition, cycling the amplification reactioncomposition to form at least one amplified product, and determining thesequence of at least part of the amplified product, thereby detectingthe RNA molecule.
 3. The method of claim 1, wherein the polypeptidecomprising double strand-specific RNA ligase activity comprises an Rnl2family ligase.
 4. The method of claim 3, wherein the polypeptidecomprising double strand-specific RNA ligase activity comprisesbacteriophage T4 RNA ligase 2 (Rnl2).
 5. The method of claim 1, whereinthe single-stranded portion of the first adaptor, the single-strandedportion of the second adaptor, or the single-stranded portion of thefirst adaptor and the single-stranded portion of the second adaptor,comprise degenerate sequences.
 6. The method of claim 1, wherein thefirst oligonucleotide comprises at least fifteen ribonucleosides.
 7. Themethod of claim 1, wherein the second adaptor comprises at least onereporter group.
 8. The method of claim 2, wherein the at least oneamplified product comprises an identification sequence.
 9. The method ofclaim 8, wherein at least one of the at least one forward primer, atleast one of the at least one reverse primer, or both, comprise anidentification sequence.
 10. The method of claim 8, wherein at least onefirst adaptor, at least one second adaptor, or at least one firstadaptor and at least one second adaptor comprises an identificationsequence.
 11. The method of claim 1, wherein the RNA molecule is a smallnon-coding RNA.
 12. The method of claim 1, wherein the sample comprisesa plurality of RNA fragments and the detecting comprises determining anexpression profile.
 13. The method of claim 12, wherein a concentrationof at least one species of RNA molecule is depleted prior to forming theligated product.
 14. The method of claim 12, wherein the samplecomprises a plurality of messenger RNA (mRNA) fragments and thedetecting comprises determining an expression profile.
 15. The method ofclaim 1, wherein the detecting comprises sequencing at least part of anamplified product, wherein the sequencing comprises a massive parallelsignature sequencing reaction, hybridizing at least one amplifiedproduct to a microarray, or cloning at least one amplified product intoa sequencing vector.
 16. The method of claim 1, wherein thesingle-stranded portion of the first adaptor, the single-strandedportion of the second adaptor, or the single-stranded portion of thefirst adaptor and the single-stranded portion of the second adaptorcomprise a sequence-specific region to selectively hybridize with acorresponding RNA molecule.
 17. A method for detecting an RNA molecule,comprising: combining the RNA molecule with at least one first adaptor,at least one second adaptor, and a double-strand specific ligase to forma ligation reaction composition, wherein the at least one first adaptorcomprises a first oligonucleotide comprising at least tworibonucleosides on the 3′-end and a second oligonucleotide thatcomprises a single-stranded portion when the first oligonucleotide andthe second oligonucleotide are hybridized together, and wherein the atleast one second adaptor comprises a third oligonucleotide thatcomprises a 5′ phosphate group and a fourth oligonucleotide thatcomprises a single-stranded portion when the third oligonucleotide andthe fourth oligonucleotide are hybridized together, ligating the atleast one first adaptor and the at least one second adaptor to the RNAmolecule to form a ligated product, wherein the first adaptor and thesecond adaptor are ligated to the RNA molecule in the same ligationreaction composition, combining the ligated product with an RNA-directedDNA polymerase, reverse transcribing the ligated product to form areverse transcribed product, combining the amplification template withat least one forward primer, at least one reverse primer, and aDNA-directed DNA polymerase to form an amplification reactioncomposition, cycling the amplification reaction composition to form anamplified product, and determining the sequence of at least part of theamplified product, thereby detecting the RNA molecule.
 18. The method ofclaim 17, wherein the single-stranded portion of the first adaptor, thesingle-stranded portion of the second adaptor, or the single-strandedportion of the first adaptor and the single-stranded portion of thesecond adaptor, comprise degenerate sequences.
 19. The method of claim17, wherein the at least two ribonucleosides of the firstoligonucleotide comprises at least fifteen ribonucleosides.
 20. Themethod of claim 17, wherein the second adaptor comprises at least onereporter group.
 21. The method of claim 17, wherein at least one of theamplified products comprises an identification sequence. 22-38.(canceled)